Dopamine receptors and genes

ABSTRACT

A mammalian D 2  dopamine receptor gene has been cloned. Thus, DNA sequences encoding all or a part of the dopamine receptor are provided, as well as the corresponding polypeptide sequences and methods for producing the same both synthetically and via expression of a corresponding sequence from a host transformed with a suitable vector carrying the corresponding DNA sequence. The various structural information provided by this invention enables the preparation of labeled or unlabeled immunospecific species, particularly antibodies, as well as nucleic acid probes labeled in conventional fashion. Pharmaceutical compositions and methods of using various products of this invention are also provided.

This application is application is a divisional of U.S. Ser. No.08/474,892, filed Jun. 7, 1995, now U.S. Pat. No. 5,880,2609, which is adivisional of U.S. Ser. No. 07/973,588, filed Nov. 9, 1992, nowabandoned, which is is a continuation of U.S. Ser. No. 07/438,544, filedNov. 20, 1989, now abandoned, which was a continuation-in-part of U.S.Ser. No. 07/273,373, filed Nov. 18, 1988, now abandoned.

This invention was made with government support under grants DK-37231and MH-45614 from the National Institutes of Health. The government hascertain rights in the invention.

BACKGROUND OF THE INVENTION

This invention relates to dopamine receptors from mammalian species andthe corresponding genes. In particular, it relates to the isolation,sequencing and/or cloning of D₂ dopamine receptor genes, the synthesisof D₂ dopamine receptors by transformed cells, and the manufacture anduse of a variety of novel products enabled by the identification andcloning of DNA encoding dopamine receptors.

Dopamine receptors in general have been implicated in a large number ofneurological and other disorders, including, for example, movementdisorders, schizophrenia, drug addiction, Parkinson's disease, Tourettesyndrome, Tardive Dyskinesia, and many others. As a result, the dopaminereceptor has been the subject of numerous pharmacological andbiochemical studies.

In general, dopamine receptors can be classified into D₁ and D₂ subtypesbased on pharmacological and biochemical characteristics (1,2). The D₂dopamine receptor has been implicated in the pathophysiology andtreatment of the mentioned disorders. In addition, it is known that theD₂ dopamine receptor interacts with guanine nucleotide binding proteinsto modulate second messenger systems (6,7).

Despite the heavy emphasis placed on elucidation of the existence andproperties of the dopamine receptor and its gene, identification,isolation and cloning have been inaccessible heretofore. This is truedespite the knowledge that other members of the family of receptors thatare coupled to G proteins share a significant similarity in primaryamino acid sequence and exhibit an archetypical topology predicted toconsist of seven putative transmembrane domains (8). Regarding theserotonin receptor, e.g., see Julius et al., Science, Vol. 241, 558(1988).

SUMMARY OF THE INVENTION

This invention has successfully identified, isolated and clonedmammalian, including human, D₂ dopamine receptor gene sequences andproduced the encoded dopamine receptor. The corresponding polypeptidehas been synthesized. Sequences of both the gene and the polypeptidehave been determined. The invention also provides a variety of new anduseful nucleic acid, cell line, vector, and polypeptide and medicinalproducts, inter alia, as well as methods of using these.

Thus, this invention relates to an isolated DNA sequence, an identifiedportion of which is a structural gene which encodes a polypeptide havingthe biological activity of a mammalian D₂ dopamine receptor. Inparticular, it relates to an isolated DNA sequence which will hybridizeto a DNA sequence encoding a mammalian D₂ dopamine receptor. It alsorelates to fragments, variants and mutants of such sequences,particularly those which also encode a polypeptide having biologicalactivity of a mammalian dopamine receptor, most particularly a mammalianD₂ dopamine receptor. In a preferred aspect, the dopamine receptor ishuman. In another preferred aspect, the sequence is that of rat D₂dopamine receptor as shown in FIG. 1. Of course, the nucleic acids ofthis invention also include complementary strands of the foregoing, aswell as sequences differing therefrom by codon degeneracy and sequenceswhich hybridize with the aforementioned sequences.

In other preferred aspects, this invention includes nucleic acidsequences and fragments useful as oligonucleotide probes, preferablylabelled with a detectable moiety such as a radioactive or biotin label.For example, such probes can hybridize with DNA encoding a polypeptidehaving the biological activity of a D₂ dopamine receptor or with DNAassociated therewith, e.g., DNA providing control of a D₂ dopaminereceptor gene or introns thereof, etc. DNA of this invention can also bepart of a vector.

The invention also involves cells transformed with vectors of thisinvention as well as methods of culturing these cells to manufacturepolypeptides, e.g., having the biological activity of a D₂ dopaminereceptor. Preferably, the cells are of mammalian origin when used insuch methods.

The invention also relates to polypeptides encoded by the foregoingnucleic acid sequences, especially to isolated mammalian dopaminereceptors, preferably of human origin. The invention further relates topolypeptides which are mutants or variants of such receptors, preferablythose wherein one or more amino acids are substituted for, inserted intoand/or deleted from the receptor, especially those mutants which retainthe biological activity of a dopamine receptor. This invention alsorelates to antibodies, preferably labelled, and most preferablymonoclonal, capable of binding a dopamine receptor amino acid sequence,preferably wherein the latter is human, or a fragment of such anantibody.

The invention further relates to compositions comprising one of thevarious products mentioned above and, typically, a pharmaceuticallyacceptable carrier as well as to methods of employing these products toachieve a wide variety of utilitarian results.

BRIEF DESCRIPTION OF THE DRAWINGS

Various other objects, features and attendant advantages of the presentinvention will be more fully appreciated as the same becomes betterunderstood when considered in conjunction with the accompanyingdrawings, and wherein:

FIGS. 1A through 1G shows the nucleotide sequence for the RGB-2 cDNA anddeduced amino acid sequence of the rat D₂ dopamine receptor. Thenucleotide sequence is numbered beginning with the first methionine ofthe long open reading frame. Nucleotide numbering appears beneath thenucleotide sequence at the right-hand end of each line. The deducedamino acid sequence is shown above the nucleotide sequence. The doubleunderline denotes the small open reading frame in the 5′ untranslatedregion. Postulated N-linked glycosylation sites are indicated byasterisks. Putative protein kinase A phosphorylation sites have a lineabove them. The intron splice junction is designated by an arrow. Thepoly A adenylation site is underlined.

FIG. 2 shows the alignment of the amino acid sequences of the rat D₂dopamine receptor, the hamster β₂-adrenergic receptor, the humanα2-adrenergic receptor, the human G-21 (serotonin 1a) receptor, theporcine muscarinic M receptor and the bovine substance K receptor. Aminoacids enclosed in solid lines and shaded represent residues that areidentical in at least two other sequences when compared to the D₂dopamine receptor. The putative transmembrane domains are indicated bybrackets labeled by Roman numerals. The numbers of residues in thevariable third cytoplasmic loop and at the C-terminus are inparentheses.

FIG. 3 shows a Northern blot analysis of RGB-2 transcripts in rat brainand pituitary. Each lane contained 20 μg of total RNA. Numbers on theright indicate molecular weight as determined from RNA size markers(BRL). A: olfactory bulb; B: hippocampus; C: cerebellum; D: posteriorcortex; E: anterior cortex; F: thalamus; G: hypothalamus; H: medulla; I:amygdala; J: mesencephalon; K: septum; L: posterior basal ganglia; M:anterior basal ganglia; N: neurointermediate lobe of pituitary; O:anterior lobe of the pituitary.

FIGS. 4A through 4C illustrates the binding of ³H-spiperone to membranesfrom L-RGB2Zem-1 cells.

FIG. 4A-1 shows saturation isotherms of the specific binding of³H-spiperone to membranes prepared from L-RGB2Zem-1 cells and ratstriatum. Results are shown from one of four independent experiments.

FIG. 4A-2 shows scatchard transformation of the data.

FIG. 4B shows competitions curves using L-RGB2Zem-1 membranes.Representative curves are shown for inhibition of specifically bound³H-spiperone by drugs in membranes from L-RGB2Zem-1 cells. Each drug wastested 3 times.

FIG. 4C is a table of K_(i) values for L-RGB2Zem-1 and rat striatum.Results are geometric means of 3 experiments in which 0.5 nM³H-spiperone was inhibited by various concentrations of unlabeled drug.For some drugs, inhibition curves in rat striatal tissue were fit bestby assuming the presence of two classes of binding sites. Theproportions of binding sites with high or low affinity for inhibitor areshown in parentheses. K_(i) values for the class of binding sitesrepresenting 10-25% of specific binding were calculated by assuming thatthe radioligand was binding to serotonin receptors with a Kd value of 1nM.

FIG. 5A shows a hydrophobicity plot of the amino acid sequence shown inFIGS. 1A through 1G; and FIG. 5B shows a hydrophobicity plot of theamino acid sequence of the β₂-adrenergic receptor. The transmembraneregions are marked by the Roman numerals.

FIGS. 6A through 6E shows a calculated restriction map of a 2477 baseEcoRI fragment of the nucleic acid sequence shown in FIG. 1.

FIGS. 7A through 7C shows a partial sequence of a human D₂ dopaminereceptor, the middle amino acid sequence shown being the correct one.

FIG. 8 shows a saturation analysis of specific [3H]spiroperidol bindingto LZR1 and LZR2 cells. Results shown are from representativeexperiments in which LZR1 and LZR2 cells were grown in control growthmedium or medium to which zinc sulfate had been added, as indicated.Data are plotted as specifically bound radioligand divided by thecorrected free concentration of radioligand (total added minus totalbound), versus specifically bound radioligand. For zinc treatments, 100μM zinc sulfate was added to growth medium for 16 hours. Both controland zinc-treated cells were then washed and grown in control medium forone day before harvesting. In the experiments shown, B_(max) valuesdetermined by nonlinear regressions analysis were 876 and 914 fmol/mg ofprotein for control LZR1 cells or LZR1 cells treated with zinc,respectively. B_(max) values for control or zinc-treated LZR2 cells were385 and 593 fmol/gm of protein, respectively.

FIGS. 9A and 9B shows the inhibition of radioligand binding by agonists.Results are plotted as specific binding, expressed as a percentage ofspecific binding in the absence of competing drug, versus the log of theconcentration of competiting drug. Membranes were prepared from LZR1cells as described in the text.

FIG. 9A shows curves from a single experiment are shown for inhibitionof the binding of [³H]spiroperidol by agonists. Each drug was testedtwice. In this experiment, the free concentration of [³ H]spiroperidolwas 230 pM, and the K_(D) value for [³H]spiroperidol was 60 pM. K_(I)values and Hill coefficients in this experiment were 5 nM and 1.05 forbromocriptine, respectively, 790 nM and 0.89 for (−)3-PPP, 8 μM and 1.0for quinpirole, 31 μM and 1.05 for (+)3-PPP, and 0.3 mM and 0.72 forLY181990.

FIG. 9B shows results are shown from one of four independent experimentsin which the effect of GTP and NaCl on the inhibition of[³H]spiroperidol binding by DA was determined. Concentrations of[³H]spiroperidol ranged from 323 to 498 pM. In this experiment, theconcentration of radioligand was 323 pM. Open circles representinhibition by DA in the presence of 0.1 mM GTP and 120 mM NaCl, whereasclosed circles represent inhibition in the absence of added GTP andNaCl. IC₅₀ values and Hill coefficients in this experiment were 29 μMand 0.65, respectively, in the absence and 115 μM and 1.03 in thepresence of GTP and NaCl.

FIGS. 10A and 10B shows the inhibition of adenylate cyclase activity inLZR1 cells. Agonists were tested for inhibition of adenylate cyclaseactivity in membranes prepared from LZR1 cells. Approximately 50 to 100μg of protein was used in each assay. Results are shown as [³²P]cAMP/mgof protein/min., expressed as a percentage of total activity in thepresence of 10 μM forskolin. Representative dose-response curves areshown for six drugs, each tested at least three times. Data are plottedas enzyme activity versus the log of the concentration of drug. No curveis plotted for the data for (−)3-PPP, since no inhibition was observed.In the experiments shown in this figure, basal and forskolin-stimulatedactivity ranged from approximately 0.8 to 1.5 and 8.5 to 15.8 pmol/mg ofprotein/min., respectively.

FIG. 11 shows the blockade of DA-sensitive adenylate cyclase. Resultsshown are means of three experiments±SE, plotted as the percentage oftotal activity in the presence of 10 μM forskolin. Forskolin (FSK) waspresent in all the experiments shown, together with 10 μM dopamine (DA)or DA and 10 μM (+)-butaclamol (BUT) as indicated. Some cells weretreated with pertussins toxin (PT) before harvesting for determinationof enzyme activity. Basal activity in control and PT-treated cells was1.2±0.07 and 1.6±0.15 pmol/mg of protein/min., respectively. Totalforskolin-stimulated activity in control cells (FSK) was 11.9±1.0pmol/mg of prptein/min. p<0.05 compared to FSK in control cells, asdetermined by a t test for paired means.

FIGS. 12A through 12C shows the reversal of dopamine inhibition bypertussis toxin pretreatment. Data presented for membrane adenylatecyclase activity represent means (x) with standard error (S.E.) and tinhibition (% Inh.) below. % Inhibition was calculated from the equation100×[1-(S-B/I-B_(I))] where B, S, and I are values of basal activity,activity in the presence of stimulator (S) or inhibitor (I),respectively and B_(I) is basal activity in the presence of inhibitor.Results were obtained from parallel assays in controls and cellspretreated (16 h, 50 ng/ml) with pertussis toxin (indicated as +P.T.).

FIG. 12A shows the results of adenylate cyclase assays. Membranes forcyclase assay were exposed acutely to 10 μM forskolin (FSK) or 100 μMdopamine (DA), and adenylate cyclase activity expressed as pmol/mgprotein/min.

(B) Intracellular cAMP. Cells were treated acutely with VIP (200 nM) anddopamine (1 μM) and cAMP accumulation (expressed as pmol/dish) wasmeasured in cell extracts.

FIG. 12C shows the results of extracellular cAMP assays. Media samplesfrom the same dishes of cells were assayed for cAMP accumulationexpressed as pmol/dish.

FIGS. 13A through 13C shows the expression of specific dopamine-D₂receptor mRNA and specific binding in GH₄ZR₇ transfectant cells.

FIG. 13A shows the results of Northern blot analysis of GH₄C₁ cell totalRNA′ (20 μg/lane). Y-axis indicate the migration of RNA molecular weightstandards (kb).

FIG. 13B-1 shows the results of transformation binding of ³H-spiperoneto membranes prepared from GH₄ZR₇ cells was characterized by saturationanalysis (see “Experimental Procedures”, Example 2). Data from one offour independent experiments are plotted as specifically boundradioligand (ordinate) versus corrected free radioligand concentration(total added minus total bound). Calculated K_(d) and B_(max) values forthis experiment were 60 pM and 1165 fmol/mg protein.

FIG. 13B-2 shows the results of transformation of the data by the methodof Scatchard which are plotted as specific bound/free (Y-axis) vs.specific bound concentrations of ³H-spiperone (X-axis).

FIG. 13C shows the results of displacement of specific ³H-spiperonebinding by dopamine: effect of GTP/NaCl. GH₄ZR₇ cell membranes wereincubated with ³H-spiperone (0.47 nM) and indicated concentrations ofdopamine (X-axis) in the absence (o) or presence (•) of 100 μM GTP/120mM NaCl. Results are shown for one of four experiments. Calculated IC₅₀and Hill coefficient values for dopamine in the experiment shown were 16μM and 0.61 in the absence of GTP/NaCl, and 56 μM and 0.85 in thepresence of GTP/NaCl.

FIGS. 14A through 14C shows the inhibition of cAMP accumulation and PRLrelease by dopamine in GH₄ZR₇ cells. Incubations were performed intriplicate as described in “Experimental Procedures”, Example 2.

FIG. 14A shows the results of assays showing inhibition of extracellularcAMP accumulation by dopamine. Parallel dishes of GH₄C₁ and GH₄ZR₇ cellswere incubated with concentrations of VIP, dopamine (D), and(−)-sulpiride (−S) of 250 nM, 10 μM and 5 μM, respectively. Untreatedcontrols are denoted as “C”. Media were collected and assayed for cAMP(ordinate) expressed as pmol/dish.

FIG. 14B shows the results of assays showing inhibition of intracellularcAMP accumulation by dopamine in GH₄ZR₇ cells. Cell extracts wereassayed for cAMP, expressed on the ordinate. Drug concentrations were asin (A), except (+)-sulpiride (+S), 5 μM.

FIG. 14C shows the results of assays showing inhibition of stimulatedPRL release by dopamine in GH4ZR₇ cells. Media samples were assayed forPRL (ordinate) after the indicated treatments. The concentrations ofVIP, TRH, dopamine (D), and (−)-sulpiride (−S) were 200 nM, 200 nM, 100nM, and 2 μM, respectively.

FIGS. 15A and 15B shows dose-response relations for dopamine inhibitionof basal and VIP-enhanced cAMP accumulation in GH₄ZR₇ cells.

FIG. 15A shows basal intra- (•) and extracellular (o) cAMP accumulationin the presence of indicated concentrations of dopamine. Basal cAMPlevels in the absence of dopamine were 22±6 pmol/dish (intracellular)and 12.4±0.6 pmol/dish (extracellular). EC₅₀ values for dopamine actionswere 4.9 nM (intracellular) and 8.5 nM (extracellular).

FIG. 15B shows VIP enhanced intra- (•) and extracellular (o) cAMPaccumulation in the presence of indicated dopamine concentrations. VIP(250 nM)-enhanced levels of intra- and extracellular cAMP (in theabsence of dopamine) were 145±1.2 pmol/dish and 146±2.8 pmol/dish, andbasal cAMP levels were 35±1.6 pmol/dish and 15±0.2 pmol/dish,respectively. EC₅₀ values for dopamine inhibition were 5.5 nM(intracellular) and 5.8 nM (extracellular).

FIG. 16 shows the specific blockade of dopamine-induced inhibition ofVIP-enhanced cAMP accumulation. GH₄ZR₇ cells were incubated in FATmedium (see “Methods”, Example 2) in the presence of 250 nM VIP and 100nM dopamine, and indicated concentrations of various dopamineantagonists, and extracellular levels of cAMP measured. Data are plottedas percent of maximal cAMP levels versus the logarithm of indicatedconcentrations of dopamine antagonists. The standard error of triplicatedeterminations was less than 8%. Basal and VIP-enhanced cAMP levels (inthe absence of dopamine and antagonists) were 25.8±1.6 pmol/dish and214±14 pmol/dish. IC₅₀ and estimated K_(i) values for antagonism ofdopamine actions were: spiperone, 0.56 mM and 30 pM; (+)-butaclamol, 4.5nM and 0.2 nM; (−)-sulpiride 38 nM and 1.8 nM; SCH23390, >1 μM;(−)-butaclamol, >10 μM. Estimated K_(I) were calculated from theequation K_(I)−IC₅₀/(1+([DA]/EC₅₀)), where [DA] is 100 nM and the EC₅₀for dopamine is 6 nM. Spiperone, (+)-butaclamol and (−)-sulpiride alonedid not alter basal cAMP levels.

FIGS. 17A and 17B shows the inhibition of adenylate cyclase bydopamine-D₂ agonists. Inhibition of adenylate cyclase activity wasassessed in the presence of 10 μM forskolin. Data are plotted as themean of triplicate assays, with enzyme activity expressed as apercentage of total activity versus the logarithm of drug concentration.Average basal adenylate cyclase activity was 4.6±0.2 pmol/mg protein/minand total forskolin-stimulated activity was 63.8±0.2 pmol/mgprotein/min. EC₅₀ values and maximal inhibition for the experimentsshown were 79 nM and 57%, respectively, for dopamine, 200 nM and 49% forquinpirole, 5 nM and 23% for bromocryptine, and 600 μM and 40% for(+)-3-PPP.

FIGS. 18A and 18J shows the nucleotide sequence of the human pituitarydopamine D₂ receptor cDNA. The deduced amino acid sequence is indicatedabove the human cDNA. Below is the nucleotide sequence of the cloned ratcDNA (see Reference 9, Example 3) and the amino acids which differbetween the two clones. Boxed regions, numbered I-VII, represent theputative transmembrane domains. Triangles indicate the exon/intrbnsplice junctions; a period is one missing base pair; asterisks identifypotential N-linked glycosylation sites and targets of protein kinase Aphosphorylation are underlined. The polyadenylation signal is doubleunderlined.

FIG. 19 shows competition curves of [3H]-domperidone binding toL-hPitD₂Zem membranes. The radioligand was used at a concentration of 1nM, and specific binding was defined using 1 μM (+) butaclamol. Data areshown for one of three experiments.

FIG. 20 shows Southern blot of human genomic DNA. The genomic BamHI1.6-kb fragment containing exon 7 was prepared from λHD2G1 and used asprobe (specific activity, 2×10⁸ cpm). DNA was digested with BamHI (lane1), BglII (lane 2), BamHI/BglII (lane 3), and HindIII (lane 4).

FIG. 21 is a schematic representation of the human dopamine D₂ receptorgene and pituitary cDNA.

(a) Restriction map of the two overlapping genomic phase, λhD2G1 andλhD2G2; A, ApaLI; B, BamHI; Bg, BglII; H, HindIII.

(b) Diagram of the human gene locus DRD₂. Exons, indicated by the boxes,are numbered. The solid boxes indicate regions of coding sequence andopen boxes, non-translated sequence. The genomic sequencing strategy isexpanded above the gene. These regions are not drawn to scale. DNAsequences (+indicates sense and −, antisense sequence) were read fromthe bottom to the top of each ladder in the directions indicated by thearrows (pointing to the right, 5′ to 3′ and to the left, 3′ to 5′). The87-bp exon is No. 5. Intron sizes were determined by Southern blottingand restriction mapping and are accurate to within 10 percent, with oneexception: intron 1 is accurate to within 20 percent.

(c) The structure of the human pituitary cDNA. Exon/intron splicejunctions are indicated by triangles. Regions encoding transmembranedomains I-VII are enclosed by boxes containing wavy lines. The 87-bpsequence is striped. The sites for translation initiation (ATG) andtermination (TAG) are indicated, as is the polyadenylation signalsequence (AATAAAA).

FIG. 22 shows a comparison of IC₅₀ values (nM) for L-hPitD2Zem,L-RGB2Zem1 and rat striatum.

FIG. 23 shows the exon/intron junctions in the human dopamine D₂receptor gene.

DISCUSSION

This invention takes advantage in a unique way of nucleotide sequencesimilarities among members of a gene family coding for receptors thatare coupled to G proteins. By using a unique hamster β₂-adrenergicreceptor (β₂AR) gene as a hybridization probe, a cDNA encoding the ratD₂ dopamine receptor was identified and isolated. The receptor has beencharacterized on the basis of three criteria: 1) the deduced amino acidsequence which reveals that it is a member of the family of Gprotein-coupled receptors, 2) the tissue distribution of thecorresponding mRNA which parallels that known for the D₂ dopaminereceptor, and 3) the pharmacological profile of Ltk− cells transformedwith the cDNA.

A rat genomic library was screened under low-stringency hybridizationconditions with a nick-translated 1.3 kb HindIII fragment containingmost of the coding region of the hamster β₂AR gene. The hamster β₂ARreceptor gene was cloned from a partial hamster lung λgt10 genomic DNAlibrary (constructed from size fractionated (5-7 kb) EcoRI digested DNA)with two oligonucleotide probes (30-mer, TCTGCTTTCAATCCCCTCATCTACTGTCGG;40-mer, CTATCTTCTGGAGCTGCCTTTTGGCCACCTGGAAGACCCT) designed from thesequence of Dixon et al. (9). The 1.3 kb HindIII fragment of the hamsterβ₂AR gene which contains most of the coding sequence of that gene waslabeled by nick translation and used to probe a rat genomic DNA libraryin the commercially available phage EMBL3. The library was transferredto Colony Plaque Screen filters (NEN) and screened with the ³²P labeledprobe using the following hybridization conditions: 25% formamide,5×SSC, 5×Denhardts, 0.1% sodium pyrophosphate, 1% SDS and salmon spermDNA (100 μg/ml) at 37° C. Filters were washed in 2×SSC and 0.1% SDS at55° C.

Several clones were found to hybridize to the hamster probe using theseconditions. One clone, called RGB-2, was found to have a 0.8 kbEcoRI-PstI fragment that hybridized to the hamster β₂AR probe inSouthern blot analysis. This fragment was sequenced and shown to have astretch of nucleotides with a high degree of identity (32 out of 40bases) to the nucleotide sequence of transmembrane domain VII of thehamster β₂AR. One of the possible reading frames demonstrated asignificant similarity to the amino acid sequence of transmembranedomains VI and VII of the hamster β₂AR. Within this genomic fragmentthere is also a 3′intron splice site (10) and 400 bp of putativeintronic sequence.

The 0.8 kb EcoRI-PstI fragment (nick translated) was used to probe a ratbrain cDNA library in λgt10 with the same hybridization conditions asabove except that 50% formamide was used. Washing of the filters wasperformed in 0.2×SSC and 0.1% SDS at 65° C. Under these high stringencyhybridization conditions, two positive clones of about 2.5 kb in sizewere identified from a library of 500,000 clones. DNA sequence wasobtained from both strands by the Sanger dideoxy chain determinationmethod using Sequenase (U.S. Biochemical) (26). Sequence and restrictionanalysis indicated that the two clones were replicas of a single cloneand contained the sequence of the genomic fragment that had been used asa probe. When the RGB-2 cDNA was used as a hybridization probe inNorthern blot analysis of mRNA isolated from rat brain, a band ofapproximately 2.5 kb was detected. This finding indicated that the RGB-2clone was nearly full length.

FIGS. 1A through 1G shows the nucleotide sequence of 2455 bases for theRGB-2 cDNA. The longest open reading frame in this cDNA codes for a 415amino acid protein (relative molecular weight (Mr=47,064)) also shown inthe figure. This molecular weight is similar to that reported for thedeglycosylated form of the D₂ dopamine receptor as determined by SDSpolyacrylamide gel electrophoresis (11). An in-frame dipeptide which is36 bases upstream from the putative initiation site is found in the 5′untranslated region of the RGB-2 cDNA. A small open reading frame hasbeen observed in the 5′ untranslated sequence of the β₂AR mRNA (9).

Several structural features of the protein deduced from the RGB-2 cDNAdemonstrate that it belongs to the family of G protein-coupledreceptors. The hydrophobicity plot of the protein sequence (FIG. 5A)shows the existence of seven stretches of hydrophobic amino acids whichcould represent seven transmembrane domains (8). Moreover, the primaryamino acid sequence of RGB-2 shows a high degree of similarity withother G protein-coupled receptors (FIG. 2). The regions of greatestamino acid identity are clustered within the putative transmembranedomains. Within these domains the RGB-2 protein has a sequence identityof 50% with the human α₂-adrenergic receptor (12), 42% with the humanG-21 receptor (13), 38% with the hamster β₂AR-adrenergic receptor (9),27% with the porcine M₁ receptor (14), and 25% with the bovine substanceK-receptor (15).

Thirdly, RGB-2 has several structural characteristics common to themembers of the family of G protein-coupled receptors. There are threeconsensus sequences for N-linked glycosylation in the N-terminus with nosignal sequence. The Asp 80 found in transmembrane domain II isconserved in all known G protein-coupled receptors. In transmembranedomain III there is Asp 114 for which a corresponding Asp residue isfound only in receptors that bind cationic amines (16). Phosphorylationhas been proposed as a means of regulating receptor function (17). Apotential site for phosphorylation by protein kinase A exists at Ser 228in the third cytoplasmic loop. In the C-terminal portion of therhodopsins and β-adrenergic receptors are found many Ser and Thrresidues which are potential substrates for receptor kinases (18). Incontrast, the short C-terminus of RGB-2 has no Ser and Thr residues.However, as is the case for the α₂-adrenergic receptor, RGB-2 has manySer and Thr residues (22 residues) in the third cytoplasmic loop whichcould serve as phosphorylation substrates. RGB-2 contains a largecytoplasmic loop (135 amino acids) between transmembrane domains V andVI with a short C-terminus (14 amino acids). This structuralorganization is similar to other receptors which are coupled by G_(i)(inhibitory G protein) such as the α₂-adrenergic receptor and the M₂muscarinic receptor. Unlike the members of the adrenergic and muscarinicreceptor families, the RGB-2 gene has at least one intron in its codingsequence which is located in transmembrane domain VI.

As a first step towards determining the identity of RGB-2, the tissuedistribution of the RGB-2 mRNA was examined by Northern blot analysis(FIG. 3).

Brain tissue was dissected from male Sprague Dawley rats and RNAisolated according to Chirgwin et al. (27) and Ullrich et al. (28). ForNorthern blotting, RNA was denatured using glyoxal and DMSO and run on1.2% agarose gels. After electrophoresis, RNA was blotted onto a nylonmembrane (N-Bond, Amersham) and baked for 2 hours at 80° C. Themembranes were prehybridized in 50% formamide, 0.2% PVP (M.W. 40,000),0.2% ficoll (M.W. 400,000), 0.05 M Tris pH 7.5, 1M NaCl, 0.1% PPi, 1%SDS and denatured salmon sperm DNA (100 μg/ml) for 16 hrs at 42° C. Arandom primed ³²P-labeled fragment (1-2 108 dpm/μg) from the 1.6 kbBamHI-BglII fragment of RGB-2 which contains the coding region of thisclone was used at 10⁷ dpm/ml in the hybridization solution from above toprobe the filters overnight at 42° C. The blots were washed twice in2×SSC and 0.1% SDS at 65° C. for 10 min., twice in 0.5×SSC and 0.1% SDSat RT for 15 min. and once in 0.1×SSC and 0.1% SDS at 65° C. for 15 min.Blots were exposed overnight at −70° C. to X-ray film with anintensifying screen.

The RGB-2 mRNA is expressed at different levels in various regions ofthe rat brain with the basal ganglia showing the highest concentration.Furthermore, the RGB-2 mRNA was found in high amounts in theneurointermediate lobe of the pituitary gland of the rat and to a lesserdegree in the anterior lobe of this gland. The expression pattern of theRGB-2 mRNA is strikingly similar to the distribution of the D₂ dopaminereceptor as determined by receptor autoradiography and binding studiesof tissue preparations (19).

In order to study the pharmacological characteristics of the receptorencoded by RGB-2, the cDNA was expressed in eucaryotic cells. The fullRGB-2 cDNA was cloned into the eucaryotic expression vector pZem3 (20)which initiates transcription from the mouse metallothionein promoter(21). This plasmid was cotransfected with the selectable neomycinphosphotransferase gene (pRSVneo) into the Ltk− mouse fibroblast cellline by the standard CaPO₄ precipitation technique (21). Cells wereselected in 350 μg/ml of G418. Transfectants were isolated and checkedfor expression of RGB-2 mRNA by Northern blot analysis. A cell lineexpressing RGB-2 designated L-RGB2Zem-1 was isolated. The RGB-2 mRNA wasnot detectable in the parent Ltk− cell line.

Since the RGB-2 mRNA displayed the tissue distribution expected of theD₂ dopamine receptor, a pharmacological study was performed of theL-RGB2Zem-1 cell line, native Ltk− cells and rat striatum using the D₂ligand ³H-spiperone.

Membranes were prepared by homogenizing cells with a Dounce homogenizerat 4° C. in 0.25 M sucrose, 25 mM Tris pH 7.4, 6 mM MgCl₂, 1 mM EDTA.The homogenizing solution was centrifuged at 800×g for 10 min. and thepellet was subjected to a second homogenization and centrifugation asbefore. The supernatants were pooled and centrifuged at 200,000×g for 1hour. The pellet of this centrifugation was resuspended in 25 mM Tris pH7.4, 6 mM MgCl₂, 1 mM EDTA at approximately 250 μg protein/ml and storedin small aliquots at −70° C. Radioligand binding assays were carried outin duplicate in a volume of 2 ml (saturation analyses) or 1 ml(inhibition curves) containing (final concentration): 50 mM Tris, pH7.4, 0.9% NaCl, 0.025% ascorbic acid, 0.001% bovine serum albumin,³H-spiperone (Amersham, 95 Ci/mmol) and appropriate drugs. In someexperiments 100 uM guanosine 5′-triphosphate was included.(+)-Butaclamol (2 uM) was used to define nonspecific binding.Incubations were initiated by the addition of 15-40 μg of protein,carried out at 37° C. for 50 minutes, and stopped by the addition of 10ml of ice-cold wash buffer (10 mM Tris, pH=7.4, and 0.9% NaCl) to eachassay. The samples were filtered through glass-fiber filters (Schleicherand Schuell No. 30) and washed with an additional 10 ml of wash buffer.The radioactivity retained on the filter was counted using a Beckman LS1701 scintillation counter. Data were analyzed as previously described(29) except that curves were drawn using the data analysis programEnzfitter. The resulting IC₅₀ values were converted to K_(i) values bythe method of Cheng and Prusoff (30).

Membranes prepared from control Ltk− cells showed no (+)-butaclamol- orsulpiride-displaceable binding of ³H-spiperone. Binding of ³H-spiperoneto membranes prepared from L-RGB2Zem-1 cells was saturable with a Kdvalue of 48 pM (FIG. 4A). This value agrees with that observed forbinding of ³H-spiperone to rat striatal membranes in parallelexperiments (52 pM). In the experiment shown in FIG. 4a, Kd and Bmaxvalues for membranes prepared from L-RGB2Zem-1 were 40 pm and 876fmol/mg of protein, whereas the corresponding values in striatalmembranes were 35 pm and 547 fmol/mg of protein. The density of bindingsites as determined in four experiments was 945 fmol/mg of protein inL-RGB2Zem-1 membranes and 454 fmol/mg of protein in rat striatalmembranes.

The binding of ³H-spiperone to membranes from L-RGB2Zem-1 cells wasinhibited by a number of drugs and the resulting K_(i) values closelymatched those obtained using striatal membranes (FIGS. 4B and 4C). TheD₂ antagonists (+)-butaclamol and haloperidol were the most potentinhibitors, followed by sulpiride. The D₁ dopamine antagonist SCH 23390and the serotonin 5HT₂ antagonist ketanserin were much less potent atblocking ³H-spiperone binding. The binding appeared to bestereoselective as (+)-butaclamol was much more potent than(−)-butaclamol at inhibiting binding. In these experiments the absoluteaffinities of dopaminergic antagonists and the rank order of potency ofthe drugs(spiperone>(+)-butaclamol>haloperidol>sulpiride>>(−)-butaclamol) agreeclosely with previously published values for the D₂ dopamine receptor(2).

All binding data for L-RGB2Zem-1 membranes were fit best by assuming thepresence of only one class of binding sites. On the other hand,inhibition by several drugs of ³H-spiperone binding to rat striatalmembranes was fit best by assuming the presence of two classes ofbinding sites. Thus, SCH 23390 and ketanserin inhibited 10-20% of³H-spiperone binding to rat striatal membranes with high affinity (FIG.4C). In rat striatal membranes, inhibition of radioligand binding bysulpiride was fit best by one class of binding sites, but 10-15% of the(+)-butaclamol-displaceable binding was not inhibited by sulpiride atthe concentrations used. It seems likely that the binding sites withhigh affinity for ketanserin and SCH 23390 and which are not displacedby sulpiride represent binding of ³H-spiperone to 5HT₂ serotoninreceptors in rat striatal membranes. Binding of SCH 23390 to 5HT₂receptors has been described previously (22). In rat striatal membranes,the apparent affinity of drugs for D₂ dopamine receptors, the class ofbinding sites comprising 80-90% of ³H-spiperone binding, wasindistinguishable from the apparent affinity of drugs to membranesprepared from L-RGB2Zem-1 cells (FIG. 4C).

The physiological effects of stimulation of D₂ dopamine receptors appearto be mediated by G_(i) (6). Inhibition of agonist binding to D₂dopamine receptors by GTP is thought to be due to GTP-induceddissociation of a receptor-G_(i) complex which has a high affinity foragonist (23,24). Although ³H-spiperone binding was inhibited by theagonist dopamine with a K_(i) of 17 um in the L-RGB2Zem-1 membranes,this dopamine binding was not responsive to the addition of GTP.However, this finding is consistent with the reported lack of G_(i) in Lcells (25). The pharmacological data presented here proves that thebinding profile of the D₂ dopamine receptor is found in Ltk− cellsexpressing the RGB-2 cDNA.

The foregoing data show that, when transfected into eucaryotic cells,the RGB-2 cDNA directs the expression of a D₂ dopamine binding protein.Since the mRNA corresponding to this cDNA is localized in tissues wherethe D₂ dopamine receptor is known to be present and since this mRNAcodes for a protein which has all the expected characteristics of a Gprotein-coupled receptor, inter alia, RGB-2 is a clone for the rat D₂dopamine receptor.

The nucleic acid sequence shown in FIGS. 1A through 1G can be insertedinto a wide variety of conventional and preferably commerciallyavailable plasmids, e.g., using EcoRI sites or other appropriate sites.See, e.g., FIG. 6 for a restriction map of the sequence of FIGS. 1Athrough 1G.

Dopamine receptor genes of this invention, particularly mammalian D₂dopamine receptor genes, based on this disclosure, can now be routinelymade, isolated and/or cloned, using many conventional techniques. Forexample, the procedure disclosed herein can be substantially reproducedfor libraries containing dopamine receptor DNA sequences.Alternatively., oligonucleotide probes can be routinely designed, e.g.,from the sequences of FIGS. 1A through 1G and/or the omitted introns,which are selective for dopamine receptor genes, especially formammalian dopamine D₂ receptor genes. These can be used to screennucleic acid libraries containing dopamine receptor nucleic acidsequences. Sequences in these libraries hybridizing to the probes,especially to all of a plurality of such probes (e.g. 2 or 3), will beDNA sequences of this invention with high probability. Of course, it isalso possible to synthesize the sequence of FIGS. 1A through 1G or anyfragment thereof using conventional methods.

This invention also enables the production of a wide variety of usefulproducts and the employment of a wide variety of useful methods, as wellas providing basic tools for the study of the regulation and function ofdopamine receptors.

These products and methods can be produced and carried out,respectively, using the well known recombinant DNA, immunochemical andother methodologies of the biotech industry. See, e.g., U.S. Pat. No.4,237,224; U.S. Pat. No. 4,264,731; U.S. Pat. No. 4,273,875; U.S. Pat.No. 4,293,652; EP 093,619; Davis et al “A Manual for GeneticEngineering, Advanced Bacterial Genetics,” Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y. (1980); Maniatis et al., MolecularCloning, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Daviset al., Basic Methods in Molecular Biology, Elsevier, N.Y. (1986);Methods in Enzymology, Berger & Kimmel (Eds.), (1987).

This invention provides, for the first time, purified and isolatedpolypeptide products having all or part of the primary amino acidsequence of the dopamine D₂ receptor as well as its biologicalproperties, including: all sites for covalent modification, such asphosphorylation and glycosylation; all primary sequence that determinesthe secondary, tertiary and quaternary structure of the functionalprotein; all parts of the molecule that provide antigenicity andantibody binding sites; all parts of the protein that provide fornoncovalent interactions with other biological molecules, such aslipids, carbohydrates, and proteins, such as guanine nucleotide-bindingregulatory proteins; all parts of the protein that make up the bindingsites for ligands, including agonists, partial agonists and antagonists;all parts of the protein that are required for functional conformationalchanges involved in normal biological activities, such asdesensitization; and all proteins that might arise as a result ofalternative splicing of the gene for this receptor.

These polypeptides can be expressed from the nucleic acids of thisinvention by procaryotic or eucaryotic hosts, e.g., bacterial, yeast ormammalian cells in culture, using fully conventional transformation ortransfection (e.g., via calcium phosphate for mammalian cells)techniques. The products of such expression in vertebrate (e.g.,mammalian and avian) cells are especially advantageous in that they areproduced free from association with other human proteins or contaminantswith which they may be associated in natural form. Preferred hosts forexpression are mammalian and include for example mouse Ltk⁻ cells,hamster CHO cells, mouse GH₄ cells, mouse C₆ cells, mouse/rat NG108-15cells and mouse AtT20 cells. For example, when the gene of FIGS. 1Athrough 1G is transfected into the commercially available growth hormoneGH₄ cells, modulation of the cAMP second messenger system has beenobserved. Preferred vectors include pzem or pRSV or viral vectors suchas vaccinia virus and retroviruses.

The polypeptides of this invention include all possible variants, e.g.,both the glycosylated and non-glycosylated forms. The particularcarbohydrates involved will depend on the mammalian or other eucaryoticcells used for the expression. The polypeptides of the invention canalso include an initial methionine amino acid residue.

Also included in this invention are polypeptides, synthetic orotherwise, duplicating the amino acid sequence of FIGS. 1A through 1Gand/or of the dopamine receptors per se of this invention, or onlypartially duplicating the same. These wholly or partially duplicativepolypeptides will preferably also retain the biological and/orimmunological activity of the dopamine receptor per se. Also includedwithin the scope of this invention are the monoclonal and polyclonalantibodies (generatable by conventional techniques and preferablylabelled) which are immunoreactive with such polypeptides.

Preferred partial polypeptides (fragments) are those including at leasta portion of the sequences located in the hydrophobic transmembranedomains V, VI and VII, shown in FIG. 2. These are the likely locationsof the ligand binding site(s), particularly domain VII. The thirdcytoplasmic loop is also an important fragment area; e.g., G-proteinbinding requires this location as well as domains V and VI. Where it isdesired to have an antibody highly specific to a particular dopaminereceptor, a fragment generating such an antibody will be selected fromthe highly unique region between transmembrane regions V and VI, i.e.,the third cytoplasmic loop, or the C-terminal domain, both of which havelow homology with other receptors, and/or the antibodies will beselected to be specific to an epitope in these regions. Anotherreceptor/gene specific region is that of the intron sequences, e.g.,those for RGB-2, mentioned above. Particularly preferred peptides whichhave been synthesized are (referring to the amino acid numbers of FIGS.1A through 1G): (A) 2-13; (B) 182-192; (C) 264-277; (D) 287-298; and (E)404-414. These are selected based on the following principles: the knownantigenicity of peptides containing a large number of Pro residuds(B/C/D); coverage of the N and C termini (A) and (E); the ability todirect antibodies towards an extracellular domain (receptor reactionregion) which will be effective to block the receptor reaction (A/C/D).Antibodies to these fragments are raised conventionally, e.g.,monoclonals by fully conventional hybridoma techniques.

This invention also relates to DNA sequences encoding the full dopaminereceptor or fragments thereof, as well as expression vectors (e.g.,viral and circular plasmid vectors) containing such whole or partialsequences. Similarly, hosts (e.g., bacterial, yeast and mammalian) orcells transformed or transfected with such vectors are also included.The corresponding methods of expressing the polypeptides correspondingto the sequences in such vectors are also included, e.g., comprisingculturing the transformed or transfected cells under appropriateconditions for large scale expression of the exogenous sequences and forthe isolation of the polypeptides as usual, e.g., from the growthmedium, cellular lysates or cellular membrane fractions.

DNA sequences of this invention also include those coding forpolypeptide variants, mutants or analogues which differ from the naturalsequence described herein. Such differences can be derived from deletionof one or more amino acid residues from the natural sequence, bysubstitution of a given such residue by another residue and/or byadditions wherein one or more amino acid residues are added to thenatural sequence. Preferably, the resultant modified polypeptide willretain at least one of the biological or immunological activities of thedopamine receptor.

Mutations likely to affect dopamine affinity activity will be those inttansmembrane domains V, VI or VII or in the third cytoplasmic loop. Inaddition, the DNA sequences also include sequences complementary to anyof the other DNA strands mentioned herein and, most notably, those shownin the Figures; DNA sequences which hybridize to the DNA sequencesdescribed herein, typically under the hybridization conditions mentionedherein or under more stringent conditions, or which hybridize tofragments of such DNA sequences; and DNA sequences which differ fromthose shown herein by the degeneracy of the genetic code. Thus, thisinvention includes all DNA sequences which encode a dopamine receptorand hybridize to one or more of the sequences shown herein. Theseinclude allelic variants as well as dopamine receptors from mammalianspecies other than the species mentioned in the experimentaldescriptions herein.

Modifications of the cDNA or genomic dopamine receptor DNA may bereadily accomplished by any of the well known techniques, includingsite-directed mutagenesis techniques. Such modified DNA sequences caninclude deletions, additions and/or substitutions made in selectedregions, e.g., not in transmembrane domains V, VI or VII or in the thirdcytoplasmic loop, where retention of the underlying biological activityof the dopamine receptor is desired.

Modified proteins which do not retain the mentioned biological activityand/or the corresponding DNA sequences will also be useful, e.g., invarious assays of this invention. In a particularly preferred suchmodification, the transmembrane domain V, VI, or VII or the thirdcytoplasmic loop will be deleted or rendered inactive, e.g., by sequencemodification. Deletion of the glycosylation sites shown in FIGS. 1Athrough 1G is also a useful variant for expression of the polypeptide,e.g., in yeast cells.

As mentioned above, it is well established that significant portions ofthe DNA sequence encoding a dopamine receptor are conserved in variousmammalian species. Consequently, using only routine experimentation, askilled worker can readily screen a DNA genomic library or, preferably,a cDNA library, e.g., from the brain of a given mammal, for the presenceof other dopamine receptor genes, especially D₂ dopamine receptor genes,using probes manufactured in accordance with the details of thesequences shown herein, including the 5′ flanking, the intronic and thestructural gene sequences shown in FIGS. 1A through 1C and the humansequence of FIGS. 7A through 7C. Probes will preferably be selected fromthe seven highly conserved transmembrane domains shown in FIG. 2,preferably domains VI and VII. Such a routine screening will identifyclones which hybridize with the probes. From these, dopamine receptorscan routinely be selected, e.g., using the techniques described herein.With respect to human D₂ dopamine receptors, particularly useful sourcesinclude, for cDNA, striatum, pituitary, neuroblastoma, kidney, placentacells, etc., and, for genomic DNA, liver, placenta cells, etc. Forprimates, e.g., rhesus monkeys, particularly useful genomic DNA or cDNAlibraries include brain, kidney and placenta cells.

With respect to human D₂ dopamine receptor genes, the partial sequenceshown in FIGS. 7A through 7C has been identified by conventionallyscreening, under the stringent hybridization conditions described abovefor the probing by the 0.8 kb EcoRI-PstI fragment of rat brain cDNA inλgt10, a pituitary cDNA library using a probe which is the full lengthrat cDNA, RGB-2. The cDNA libraries mentioned herein were prepared byfully conventional methods, e.g., as described in the references citedabove, e.g., Davis et al. This sequence or fragments thereof can also beuseful as a probe, for example, to screen conventional libraries asmentioned above for human dopamine receptor genes in accordance with theforegoing and other fully conventional procedures. As can be seen bycomparing the sequence of FIGS. 7A through 7C with the sequences shownin FIGS. 1A through 7C above, the partial human sequence of FIGS. 7Athrough 7C has high homology with RGB-2 beginning at amino acid no. 259of FIG. 1.

Similarly, this invention more generally includes mammalian D₂ dopaminereceptor genes in the broadest sense, e.g., both regulatory andstructural such genes, alone or in combination, e.g., in reading frame.For example, using the routine methods discussed herein, such genes havebeen and can be cloned from mammalian DNA libraries. As well, thisinvention includes biologically active fragments of such genes, e.g.,fragments encoding polypeptides having the biological activity of amammalian D₂ dopamine receptor, or fragments useful in controllingexpression of such encoding fragments, or fragments useful as probes forany such gene or fragment, e.g., by hybridizing therewith.

The various polypeptides and sequences of this invention may beconventionally labeled with detectable marker substances, typically bycovalent association, and further typically by radiolabeling, or in thecase of DNA, with non-isotopic labels such as biotin. The polypeptideproducts (e.g., labelled antibodies) can be used conventionally todetect and quantitate the presence of dopamine receptors in varioussamples; the DNA-labeled products can be conventionally employed in theusual hybridization methods (e.g., Northern blots, Southern blots, spotassays, etc.) to detect and quantitate the presence of associatednucleic acids (DNA, RNA) in samples, e.g., to locate the dopamine genepositions in various mammalian chromosomal maps, to determine whethermRNA or receptor concentrations are abnormally high or low in comparisonto standard levels, etc. They will also be useful, again using fullyconventional prodedures, to identify dopamine receptor gene disorders(defective or aberrant genes) in in vitro diagnostic procedures on DNAsamples from given patients, e.g., to detect chromosomal defects, e.g.,using RFLP analyses (see, e.g., Genes III, Levin, John Wiley and Sons(1987). For example, since dopamine receptors have been implicated inschizophrenia, products and methods of this invention can be used tocharacterize nucleic acids, e.g., in size fractionated form, fromschizophrenia patients and, inter alia, classify patients according toschizophrenia subgroups, make diagnoses based on comparisons to standardDNA fractionation arrays, etc. Of course, they can also be used, e.g.,in the mapping of the human or other mammalian genome, as gene markersto identify accompanying genes, e.g., in RFLP analyses, and, whereapplicable, other disorders. The gene-unique regions discussed above,e.g., the intron regions, the third cytoplasmic loop, etc., will beespecially useful in this regard.

Typical assays in which the polypeptides of this invention can beutilized include any of the well known immunoassay techniques such asRIA, ELISA, etc., both of in vitro and in vivo nature. Various fragmentsof the polypeptide sequence of the dopanine receptor can also beutilized conventionally for producing corresponding polyclonalantibodies or preferably monoclonal antibodies (e.g., by conventionalpreparation and expression of corresponding hybridomas) for epitopeswithin the given fragment. The antibodies will often be conventionallylabelled, e.g., radio- or enzymatically labelled. In a preferred aspect,the resultant polyclonal or monoclonal antibodies will also beimmunospecific with respect to not only the mentioned fragment, but alsothe full protein. Such antibodies will be conventionally employable, forexample, in the detection and affinity purification or chromatography ofdopamine receptor and related products.

Of course, the polypeptides of this invention include those expressed inaccordance with conventional procedures from cells as mentioned above,as well as those which are synthetically prepared also usingconventional procedures. This invention enables for the first timenon-natural-preparation of mammalian dopamine receptors substantiallyfree of constituents of their natural environment. It is in this sensethe term “substantially pure” is used herein, i.e., substantially freeof these natural constituents. The invention also includes DNA sequenceswhich can be isolated from the various sources mentioned above orsynthetically prepared using fully conventional methods.

Also included within the scope of this invention are pharmaceuticalcompositions comprising effective amounts of one or more of thepolypeptide products of this invention or one or more of the nucleicacid sequences of this invention, in admixture with suitableconventional diluents, adjuvants and/or carriers well known in thepharmaceutical industry. These can be utilized for in vitro uses, e.g.,for detection of the presence of a dopamine receptor in a sample or ofthe presence of a gene or an abnormal gene in a sample or for increasingthe concentration of receptor or its gene in a sample, or for in vivouses such as gene therapy (e.g., to render a defective gene or geneproduct inactive, e.g., block it by an appropriate monoclonal antibody)and/or to provide new functional genes (e.g., using retroviralvectors)), or to provide an increased concentration of dopamine receptorin a given location, or to modulate receptor expression and/or activity,e.g., by administration of antisense oligosequences, all in mammalsincluding humans. Thus, these compositions will be useful to treatdisease conditions, inter alia, those associated with abnormalities inthe structure, expression or concentration of the dopamine receptor orits gene, such as those mentioned in the foregoing. Specific effectivedosages for a given condition in a given patient will vary, as is wellknown, with the usual conditions, including the overall condition of thepatient, body weight, the identity and severity of the particulardopamine-deficiency disease state, etc.

The polypeptides of this invention can also be used in, e.g.,competitive binding assays, to test for the affinity thereto ofcandidate chemical substances such as drugs, e.g., affinity (e.g.,agonistic or antagonistic) to D₂ dopamine receptors. Such procedures canbe carried out, e.g., as pharmaceutical screening tests, using fullyconventional procedures, analogous to those described herein and/or toknown protocols based on natural sources of dopamine receptors, e.g.,analogous to known tests for inhibition of the binding of tritiateddopamine agonists and antagonists to striatal receptors per the methodsof Schwarcz et al., J. Neurochemistry, 34 (1980), 772-778 and Creese etal., European J. Pharmacol., 46 (1977), 377-381, and to those for otherreceptors. It is also possible to screen substances for ability tomodify or initiate a response which is triggered by ligand binding to adopamine receptor, e.g., cellular responses such as modulation of secondmessenger systems. Such analyses can utilize cells of this inventiontransformed with nucleic acid sequences of this invention.

Antibodies of this invention to the dopamine receptors or other regionsof dopamine receptor genes, especially the D₂ receptor, can also be usedin diagnostic imaging techniques, e.g., by radiodiagnostic, MRI orpositron imaging. Radio-, paramagnetic- or positron-labels can beconventionally attached to the antibodies (preferably monoclonal innature), e.g., via covalent bonding to chelating groups for a positronemitting, radionucleotide or paramagnetic metal. MRI, radiosensitive orpositron imaging can then be effected with these agents usingconventional methods. See, e.g., Maziere et al., Life Sci., 35, 1349(1984).

Suitable pharmaceutical carriers include water, saline, human serumalbumin, etc. The compositions can also include other active ingredientssuitable for amelioration of the particular disease state involved,e.g., conventional dopamine agonists, dopamine antagonists, etc. Thecomponents of this invention can be provided in conventional kit formcontaining, e.g., an antibody or a DNA probe (e.g., able to detect genehomologies or anomalies) along with detection method-specific reagentssuch as enzymes, substrates, materials for analyzing DNA restrictionfragments, etc.

The DNA sequences of this invention are also useful to prepare thecorresponding transgenic animals, in particular nonhuman mammals, e.g.,rats, monkeys, etc., using known methods, e.g., analogous to thosedescribed in U.S. Pat. No. 4,736,866. Such animals, e.g., areparticularly useful for commercial research purposes. The DNA sequencesor the corresponding mRNA can also be used conventionally to injectoocytes, e.g., from frogs, which can then be conventionally used inbinding or second messenger analyses. Moreover, the availability of theprimary amino acid sequence itself enables experimental andcomputational modeling and understanding of the secondary and tertiarystructures of the dopamine receptor. This three-dimensional informationprovides a basis for modeling and understanding the details of thereceptor function, e.g., interaction with the cell membrane, its ligand(binding pocket), associated proteins, messenger systems, etc. Suchanalyses enable rational drug design whereby, e.g., new dopaminereceptor affecting chemical agents can be designed in accordance withthe details of this interaction.

NUMBERED REFERENCES FOR BACKGROUND, SUMMARY, FIGS. 1-7, AND DISCUSSION(Excluding Examples)

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Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The mentioned embodiments are, therefore, to beconstrued as merely illustrative and not limitative of the remainder ofthe disclosure in any way whatsoever.

From the foregoing description, one skilled in the art can easilyascertain the essential characteristics of this invention and, withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions.

The entire texts of all applications, patents and publications citedabove are hereby incorporated by reference.

EXAMPLES Example 1

Summary

We recently cloned a complementary DNA for the rat dopamine D-2receptor, making it possible to create cell lines expressing thisreceptor. A cell line (LZR1) was created by transfecting the D-2 cDNA(RGB-2) into mouse fibroblast Ltk⁻ cells. LZR1 cells, previouslydescribed as L-RGB2Zem-1 cells (1), expressed a high density of D-2receptors, whereas the wild-type cells did not. Although transcriptionof the RGB-2 cDNA is regulated by the zinc-inducible mousemetallothionein promoter, the density of D-2 receptors on membranesprepared from LZR1 cells was not increased after the cells were treatedwith zinc. A second cell line derived from Ltk⁻ cells, designated LZR2,had a lower density of D-2 receptors in the uninduced state, andtreatment with zinc induced a 50% increase in the receptor density. Anumber of agonists competitively and stereoselectively inhibited thebinding of [³H]spiroperidol to the expressed D-2 receptors. In LZR1cells, dopamine was a more potent inhibitor of radioligand binding inthe absence than in the presence of GTP and NaCl. Dopamine reducedforskolin-stimulated adenylate cyclase activity by 27% in membranesprepared from LZR1 cells. Inhibition by dopamine was blocked by(+)-butaclamol or prior treatment of intact cells with pertussis toxin.These data indicate that the RGB-2 cDNA directs the expression of adopamine D-2 receptor capable of interacting with guaninenucleotide-binding proteins and inhibiting adenylate cyclase activity.Furthermore, the RGB-2 cDNA provides a means of creating many cell linesthat will be useful tools for the biochemical and pharmacologicalcharacterization of dopamine D-2 receptors.

Introduction

Dopamine (DA) receptors have been classified into two subtypes based onfunctional and pharmacological profiles (2). DA D-2 receptors arecharacterized functionally by their ability to inhibit adenylate cyclaseactivity (3). Activation of D-2 receptors also inhibits calcium channels(4,5), increases potassium conductance (6), and may inhibit accumulationof inositol phosphates (7,8). One factor that has impeded research onthe regulation and functional characteristics of DA receptors has beenthe lack of cell lines that express the receptors. One cell line,derived from a prolactin-secreting tumor, has recently been described inwhich DA inhibits adenylate cyclase activity and prolactin secretion(9).

We recently cloned a rat brain complementary DNA (cDNA), designatedRGB-2, that has significant homology with β₂-adrenergic receptors andother receptors that interact with guanine nucleotide-binding proteins.Three lines of evidence indicate that the RGB-2 cDNA encodes the DA D-2receptor: (1) The deduced amino acid-sequence of the protein suggeststhe existence of the seven membrane-spanning domains typical ofreceptors coupled to guanine nucleotide-binding proteins (10); (2) thedistribution of messenger RNA that hybridizes with the cDNA parallelsthe distribution of the D-2 receptor; and (3) when the RGB-2 cDNA istransfected into cells that lack high affinity binding of the D-2selective ligand [³H]spiroperidol, the cells express binding sites forthe radioligand with a pharmacological profile characteristic of D-2receptors (1).

The cloning of a D-2 receptor cDNA makes it possible to express DAreceptors in cell lines in which the effects of receptor activation canreadily be determined. We previously described the binding of[³H]spiroperidol and other D-2 antagonists to a line of cells derived bytransfection of mouse L cells with the RGB-2 cDNA under the control of azinc-inducible mouse metallothionein promoter (1). We also reportedthat, under the assay conditions used previously, the binding of DA toLZR1 membranes was not responsive to GTP. We now demonstrate that theD-2 receptor encoded by the RGB-2 cDNA is functional with respect to theguanine nucleotide sensitivity of the binding of agonists and theability of agonists to inhibit adenylate cyclase activity.

Methods

Materials: [α-³²P]Adenosine 5′-triphosphate (ATP, 10-50 Ci/mmol) and[³H]-cyclic AMP (31.9 Ci/mmol) were purchased from New England Nuclear(Boston, Mass.), and [³H]spiroperidol (95 Ci/mmol) was purchased fromAmersham (Arlington Heights, Ill.). Guanosine 5′-triphosphate (GTP), DA,cyclic μMP, 3-isobutyl-1-methyl-xanthine, ATP, andyforskolin werepurchased from Sigma Chemical Company (St. Louis, Mo.). Quinpirole,LY181990 (Lilly Laboratories), bromocriptine (Sandoz ResearchInstitute), and (+) and (−)3-PPP (Astra) were generous donations.

Transfection: The full RGB-2 cDNA was cloned into the plasmid pZem3(11). The cDNA and the vector were made compatible by partially fillingin the Bgl II site on the vector and a Sal I site on the cDNA adaptor.This plasmid was co-transfected with the plasmid pRSVneo into mouse Ltk⁻cells by a CaPO₄ precipitation technique (12). Transfectants wereselected in 350 μg/ml of G418, isolated, and screened for expression ofRGB-2 mRNA by Northern blot analysis. The subclone LZR1, selected on thebasis of high expression of RGB-2 mRNA, was partially characterizedpreviously as L-RGB2Zem-1 (1). A second cell line, LZR2, was isolated inthe same way.

Tissue culture: Cells were plated at a density of 20,000 cells/cm² in150 mm diameter Falcon tissue culture plates (Beckton Dickinson, LincolnPark, N.J.), subcultured by replacing the growth medium withtrypsin-EDTA (0.1% trypsin, 0.02% EDTA in phosphate-buffered saline) orfed on day 3, and harvested on day 5 or 6. Cells were grown inDulbecco's modified Eagles' medium (Sigma), supplemented with 5% fetalbovine serum and 5% iron-supplemented calf bovine serum (Hyclone, Logan,Utah), in an atmosphere of 10% CO₂/90% air at 37°. Cells were lysed byreplacing the growth medium with ice-cold hypotonic buffer (1 mMNa⁺-HEPES, pH 7.4, 2 mM EDTA). After swelling for 10-15 min, the cellswere scraped from the plate and centrifuged at 24.000×g for 20 min. Theresulting crude membrane fraction was resuspended with a BrinkmannPolytron homogenizer at setting 6 for 10 sec in Tris-isosaline (50 mMTris-HCl, pH 7.4, and 0.9% NaCl) and stored at −70° for receptor bindingexperiments or resuspended in Tris-isosaline, centrifuged again at24,000×g for 20 min., and resuspended in Tris-isosaline for immediateuse in adenylate cyclase experiments.

Receptor binding assay: The membrane preparation was thawed, centrifugedat 24,000×g for 20 min., and resuspended in Tris-isosaline except whereindicated. Aliquots of the membrane preparation were added to assaytubes containing (final concentrations) 50 mM Tris-HCl, pH 7.4, 0.9%NaCl, 0.025% ascorbic acid, 0.001% bovine serum albumin,[³H]spiroperidol, and appropriate drugs. (+)-Butaclamol (2 μM) was usedto define nonspecific binding, which was typically less than 10% oftotal binding at concentrations of radioligand near the K_(D) value.Assays were carried out in duplicate in a volume of 2 ml for saturationanalyses or 1 ml for inhibition analyses. Incubations were initiated bythe addition of 15-50 μg of protein, carried out at 37° for 50 min., andstopped by the addition of 10 ml of ice-cold wash buffer (10 mM Tris, pH7.4, and 0.9% NaCl) to each assay. The samples were filtered throughglass-fibre filters (Schleicher & Schuell No. 30) and washed with anadditional 10 ml of wash buffer. The radioactivity retained on thefilters was counted using a Beckman LS 1701 scintillation counter. Datawere analyzed by nonlinear regression using the data analysis programEnzfitter (Elsevier-Biosoft). In competition experiments, K_(I) valueswere calculated from experimentally determined IC₅₀ values by the methodof Cheng and Prusoff (13). Averages for K_(I) and K_(D) values are thegeometric means. In experiments designed to assess the effect of GTP andNaCl on the binding of DA, fresh tissue was used. Cells were harvested,centrifuged, and Mg²⁺ resuspended in Tris-Mg²⁺ (50 mM Tris, pH 7.4, and4 mM MgCl₂). Tissue was incubated for 15 min. at 37° in this bufferbefore re-centrifugation. The resuspended protein was added to assayscontaining Tris-Mg²⁺ with no added NaCl or GTP, or Tris-Mg²⁺ with 120 mMNaCl and 100 μM GTP.

Adenylate cyclase assay: The conversion of [α³²P]ATP to [³²P]cAMP wasdetermined essentially as described by Salomon et al. (14). Membranes(50-100 μg of protein) resuspended in Tris-isosaline were added in avolume of 0.1 ml to an assay of 0.2 ml containing 50 mM Tris-HCl, pH7.4, 5 mM cAMP, 1 mM 3-isobutyl-1-methylxanthine 1 mM MgCl₂, 0.5 mMEGTA, 0.25 mM ATP, 30 μM GTP, approximately 2×10⁶ cpm of [α-³²P]ATP, andvarious drugs. Assays were initiated by warming to 25° and terminatedafter 20 minutes by cooling to 0°, then adding trichloroacetic acid (100μl of a 30% solution) to each assay. [³H]Cyclic AMP (approximately30,000 cpm) was added to each assay as an internal standard. The assayvolume was brought up to 1 ml with water, and tubes were centrifuged for10 min. at 2000×g. Cyclic AMP in the supernatant was isolated bysequential chromatography on columns containing Dowex AG50W-X4 resin andneutral alumina. The 2-ml eluate from each column of alumina wasdissolved in 10 ml of Bio-Safe II (RPI, Mount Prospect, Ill.) for liquidscintillation counting. Dose-response curves for inhibition of adenylatecyclase activity by agonists were analyzed by nonlinear regression usingthe program Enzfitter. The data were fit to the equation:

E=(100−E_(max))/(1+(A/EC₅₀)^(N))+E_(max)

where E is the amount of enzyme activity, expressed as percentage oftotal stimulated activity, A is the concentration of agonists, EC₅₀ isthe concentration of agonist causing half-maximal inhibition of enzymeactivity, E_(max) is the enzyme activity observed in the presence ofmaximally inhibiting concentrations of agonist, expressed as thepercentage of total stimulated activity, and N is a slope factor.Averages of EC₅₀ values are the geometric means. Protein concentrationwas determined by the method of Peterson (15).

Results

Saturation analysis of the binding of [³H]spiroperidol: The density ofD-2 receptors on membranes prepared from LZR1 cells was determined bysaturation analysis of the binding of [³H]spiroperidol (FIG. 8). Sincethe RGB-2 cDNA is under the control of the zinc-inducible mousemetallothionein promoter, the effect of prior treatment of cells with100 μM zinc sulfate on the binding of [³H]spiroperidol was alsodetermined. The density of binding sites was 736±140 fmol/mg of proteinin control LZR1 cells, and 759±155 fmol/mg of protein in LZR1 cellstreated with zinc (n=2). A second cell line, designated LZR2, had alower density of D-2 receptors (mean B_(max)±SE, 435±71 fmol/mg ofprotein). In contrast to LZR1 cells, the density of binding sites onLZR2 cells was increased 50% by zinc treatment to 630±52 fmol/mg ofprotein. The mean K_(D) value of 42 pM for control LZR1 and LZR2 cells(pK_(D)±SE, 10.37±0.15, n=4) did not differ significantly from the mean; value of 47 pM for zinc-treated cells (10.33±0.17). Scatchardtransformation of saturation analyses from all experiments yieldedstraight lines. Wild-type Ltk⁻ cells had no detectable displaceablebinding of [³H]spiroperidol (data not shown). Since LZR1 cells had thehigher density of D-2 receptors, these cells were used in all subsequentexperiments.

Inhibition of radiolipand binding by agonists: The apparent affinity ofD-2 receptors for several agonists and related compounds was determinedin two experiments (FIG. 9A, Table 1). of the six compounds tested,bromocriptine was the most potent with a mean K_(I) value of 2 nM,whereas LY181990, the inactive enantiomer of the D-2-selective agonistquinpirole, was the least potent. All assays were carried out in thepresence of 0.1 mM GTP and 120 mM NaCl, using membranes prepared fromLZR1 cells.

In other experiments, the effect of GTP and NaCl on inhibition ofradioliqand binding by DA was determined (FIG. 9B). Freshly preparedmembranes were used for these experiments, and 4 mM MgCl₂ was added tothe homogenization and assay buffers. In the absence of GTP and NaCl,the mean IC₅₀ was 25 μM [mean−log(IC₅₀)±SE=4.6±0.3]. Addition of GTP andNaCl to the assay buffer increased the mean IC₅₀ value to 167 μM(3.8±0.3, n=4), without altering the binding of [³H]spiroperidol. Hillcoefficients in the absence of GTP and NaCl ranged from 0.56 to 0.68(mean=0.64) whereas in the presence of GTP and NaCl values ranged from0.77 to 1.1 (mean=0.94). In the absence of GTP and NaCl, inhibitioncurves for DA could be fit best by assuming the presence of two classesof binding sites. One class of high affinity sites, representing 48±14%of the total number of receptors, had a mean K_(I) for DA of 0.3 μM. Thesecond class, representing 52±13% of the total number of receptors had amean K_(I) value of 24 μM.

Inhibition of adenylate cyclase activity by agonists: In freshlyprepared membranes from LZR1 cells, but not in membranes from wild-typeLtk⁻ cells, DA caused a concentration-dependent attenuation offorskolin-stimulated adenylate cyclase activity. Maximal inhibition was27% of total activity, with an EC₅₀ value of 624 nM (FIG. 10A; Table 1).Quinpirole was approximately as efficacious as dopamine; that is, themaximal inhibition induced by quinpirole was similar to that induced byDA. LY181990 was less potent and less efficacious than its active isomer(FIG. 10B; Table 1), indicating that D-2 receptor-mediated inhibition ofadenylate cyclase activity was stereoselective. The finding thatLY181990 caused measurable inhibition of enzyme activity in only 2 outof 3 experiments (Table 1) suggests that the compound has little or noagonist activity. Similarly, (−)3-PPP did not have detectable agonistactivity. Bromocriptine, with an EC₅₀ value of 45 nM, was the mostpotent agonist. Bromocriptine and (+)3-PPP were less efficacious thanDA; thus, the drugs appeared to be partial agonists.

Inhibition of adenylate cyclase activity in LZR1 cells by 10 μM DA wasprevented by including 10 μM (+)-butaclamol in the assay (FIG. 11),indicating that inhibition of DA is receptor-mediated. Also, treatmentof LZR1 cells with pertussis toxin (50 ng/ml of growth medium for 16hours) blocked DA-inhibited enzyme activity in membranes prepared fromthe cells (FIG. 10C), suggesting that G_(i) mediates inhibition ofenzyme activity by DA. Interestingly, forskolin-stimulated adenylatecyclase activity in membranes from pertussis toxin-treated cells wasapproximately 2.5-fold greater than activity in control membranes.

TABLE 1

Inhibition of Radioligand Binding and Adenylate Cyclase Activity byAgonists

The apparent affinity (K_(I)) of 6 drugs for D-2 receptors on membranesprepared from LZR1 cells, determined by inhibition of the binding of[³H]spiroperidol (0.2 nM), is shown, as well as the concentration ofeach drug that caused half-maximal inhibition of forskolin-stimulatedadenylate cyclase activity (EC₅₀). Values for drug concentrations,expressed as μM, are the geometric means of results from 3 experiments(EC₅₀, quinpirole and (+)3-PPP), 4 experiments (EC₅₀-DA) or 2 (K_(I),all drugs; EC₅₀, bromocriptine) experiments. The maximal inhibition ofadenylate cyclase activity observed (Max) is expressed as the mean±SEMof the percent inhibition of total activity in the presence of 10 μMforskolin. For LY181990, the results shown are from two experiments inwhich inhibition of enzyme was observed. There was no inhibition in athird experiment.

Drug K_(I) EC₅₀ Max Dopamine 17 0.6  27 ± 3% Quinopirole 9 0.7  28 ± 2%LY181990 277 5.0  10 ± 2% Bromocriptine 0.0024 0.04 17 ± 1% (+)3-PPP 334.0  16 ± 3% (−)3-PPP 0.87 — —

Discussion

As reported previously, Ltk⁻ cells transfected with a rat D-2 receptorcDNA express a high density of DA D-2 receptors (1). We havecharacterized one subclone of these cells, designated LZR1, that stablyexpresses D-2 receptors at a density of 750 to 1000 fmol/mg of protein(present results; ref. 1).

As the D-2 cDNA is contained in the plasmid pZem3, under the regulationof a zinc-inducible promoter, the effect of zinc treatment on theproperties of radioligand binding was determined. D-2 receptors on LZR1cells appear to be maximally expressed in the absence of zinc, so thatzinc treatment caused no increase in the density of receptors. The lackof responsiveness to zinc is not a characteristic of the LtK⁻ cell line,since we have isolated a second transfected line of Ltk⁻ cells, LZR2, inwhich the density of D-2 receptors is elevated approximately 50% bytreatment with zinc, from 435 to 630 fmol/mg of protein. The affinity ofD-2 receptors for [³H]spiroperidol was not significantly altered by zinctreatment.

The apparent affinity of D-2 receptors on LZR1 cells for severalagonists and related drugs was determined by inhibiting the specificbinding of [³H]spiroperidol with the drugs in the presence of GTP.Bromocriptine was an extremely potent agonist, with a K_(I) value of 2nM. The potency of quinpirole, like that of DA, was approximately 10 μM.The binding of agonists was stereoselective, since LY181990 was muchless potent than its active enantiomer, quinpirole, and (−)3-PPP wasmore potent than (+)3-PPP. These data extend our previous investigationof the binding of antagonists to D-2 receptors on LZR1 cells (1).

Physiological effects of stimulation of D-2 receptors appear to bemediated by a guanine nucleotide-binding protein, G_(i), that inhibitsadenylate cyclase activity (16). High affinity binding of agonists isthought to represent a ternary complex composed of agonist, receptor,and the α-subunit of G_(i) (G_(iα)), and inhibition of agonist bindingto D-2 receptors by GTP represents GTP-induced uncoupling of D-2receptors from G_(iα). To evaluate the ability of D-2 receptors encodedby the RGB-2 cDNA to couple to G proteins, the effect of GTP on thepotency of DA for inhibition of radioligand binding was determined. Inpreliminary studies using LZR1 cells, we found that in our standardassay buffer containing 120 mM NaCl and no added Mg²⁺, the binding of DAwas not sensitive to GTP (1), although under the same conditions thebinding of DA to rat striatal membranes is inhibited by GTP (data notshown). There were three possible explanations for the lack ofsensitivity to GTP in LZR1 membranes: (1) LZR1 cells, derived from Ltk⁻cells, could lack the appropriate G-protein. (2) The RGB-2 cDNA couldencode only a binding subunit of the D-2 receptor. This possibilityseemed unlikely because of the similarity between the predicted primarystructure of the protein encoded by RGB-2 and other receptors coupled toguanine nucleotide-binding proteins. (3) It could be that the ionicconditions of the binding assay were not appropriate for formation ordestabilization of the ternary complex. In the studies described here,ionic conditions were varied to increase the likelihood of observingGTP-sensitive binding. To maximize high-affinity agonist binding, tissuewas preincubated with MgCl₂, and MgCl₂ was included in the assay buffer(17). To maximize GTP-induced destabilization of the ternary complex,NaCl was added together with GTP (17). Under these conditions, additionof GTP and NaCl decreased the potency of DA for D-2 receptors andincreased the slope of the inhibition curve in membranes from LZR1cells. It is interesting that the ionic requirements for formation anddestabilization of the ternary complex seem to be more stringent inmembranes from LZR cells than in membranes from rat striatum.

Inhibition of adenylate cyclase activity by DA D-2 receptors is awell-characterized phenomenon (3, 18, 19). Inhibition of adenylatecyclase activity by several drugs was assessed in membranes from LZR1cells. Maximally effective concentrations of DA and the D-2-selectiveagonist quinpirole decreased forskolin-stimulated enzyme activity byalmost 30%. Inhibition of enzyme activity by DA was blocked by the D-2antagonist (+)-butaclamol. The most potent agonist tested,bromocriptine, appeared to be a partial agonist, as reported by others(19, 20). Inhibition of adenylate cyclase by agonists wasstereoselective, since LY181990, the dextrorotatory enantiomer ofquinpirole, had little or no efficacy. As has been reported previously,the partial agonist (+)3-PPP is a stronger agonist than (−)3-PPP,although (−)3-PPP binds to D-2 receptors with higher affinity (21, 22).We observed no inhibition of adenylate cyclase activity by (−)3-PPP inmembranes from LZR1 cells. The EC₅₀ values determined for inhibition ofadenylate cyclase activity by agonists were generally lower than theK_(I) values determined in assays of ligand binding (Table 1). Thiscould be due to the presence of a receptor reserve on these cells,although the observation that drugs differed in maximal inhibition ofenzyme activity suggests that there is not a large receptor reserve evenfor the most efficacious agonists, DA and quinpirole. Also, there wouldbe no receptor reserve for a partial agonist such as (+)3-PPP. Analternative explanation is that inhibition of adenylate cyclase activitycould be related to the binding of agonists to D-2 receptors in ahigh-affinity state induced by formation of the ternary complex, as hadbeen proposed for inhibition of enzyme activity by DA in the anteriorpituitary (23). On the other hand, K_(I) values in the present report,determined in the presence of GTP and NaCl, reflect the binding ofagonists to receptors in a state of low affinity. Direct comparisons aredifficult, since adenylate cyclase and radioligand binding assays werecarried out at different temperatures, but the K_(I) value for DAbinding to the high-affinity class of sites (0.3 μM) is close to theEC₅₀ value for DA-inhibited adenylate cyclase (0.6 μM), whereas theK_(I) values for the binding of DA to the low-affinity class of sites(24 μM) and binding in the presence of GTP (17 μM) are considerablyhigher.

DA did not inhibit adenylate cyclase activity in membranes from LZR1cells that had been treated with pertussis toxin. Since pertussistoxin-catalyzed ADP-ribosylation of G_(i) prevents G_(i)-mediatedinhibition of adenylate cyclase, this finding is consistent with thehypothesis that D-2 receptors interact with G_(iα) in the transfectedLZR1 cells. As has been observed for stimulation of adenylate cyclaseactivity by isoproterenol after pertussis toxin-treatment of other celltypes (24), treatment of intact LZR1 cells with pertussis toxinpotentiated the ability of forskolin to stimulate adenylate cyclaseactivity, suggesting that in some cell lines G. normally acts toattenuate forskolin- and hormone-stimulated adenylate cyclase activity.

We have characterized a cell line, transfected with the RGB-2 cDNA, thatstably expresses a high density of D-2 receptors. With this cell line,it was determined the cDNA encodes a DA D-2 receptor that interactsproductively with a guanine nucleotide-binding protein to inhibitadenylate cyclase activity. It seems likely that the RGB-2 cDNA woulddirect the expression of a functional D-2 receptor in almost any type ofcell. For example, GH₄C₁ cells, derived from a rat pituitary tumor (25),are prolactin-secreting cells that lack DA receptors, even thoughlactrotrophs in the rat anterior pituitary express D-2 receptors.Transfection of the RGB-2 cDNA into GH₄C₁ cells results in theexpression of a D-2 receptor with functional characteristics similar tothose described here (26). Cell lines created by transfection with a D-2receptor cDNA will be useful in the study of mechanisms of action andregulation of D-2 receptors.

References for Example 1

1. Bunzow, J. R., H. H. M. Van Tol, D. K. Grandy, P.Albert, J. Salon, M.Christie, C. A. Machida, K. A. Neve, and O. Civelli. Cloning andexpressing of a rat D₂ dopamine receptor cDNA. Nature (Lond.)336:783-787 (1988).

2. Kebabian, J. W., and D. B. Calne. Multiple receptors for dopamine.Nature (Lond.) 277:93-96 (1979).

3. De Camilli, P., D. Macconi, and A. Spada. Dopamine inhibits adenylatecyclase in human prolactin-secreting pituitary adenomas. Nature (Lond.)278:252-254 (179).

4. Malgaroli, A., L. Vallar, F. R. Elahi, T. Pozzan, A. Spada, and J.Meldoiesi. Dopamine inhibits cytosolic Ca²-increases in rat lactotrophcells: Evidence of a dual mechanism of action. J. Biol. Chem.262:13920-13927 (1987).

5. Drouva, S. V., E. Rerat, C. Bihoreau, E. Laplante, R.Rasolonjanahary, H. Clauser, and C. Kordon. Dihydropyridine-sensitivecalcium channel activity related to prolactin, growth hormone, andluteinizing hormone release from anterior pituitary cells in culture.Interactions with somatostatin, dopamine, and estrogens. Endocrinology123:2762-2773 (1988).

6. Lacey, M. G., N. B. Mercuri, and R. A. North. Dopamine acts on D₂receptors to increase potassium conductance in neurones of the rabsubstantia nigra zona compacta. J.Physiol. (Lond.) 392:397-416 (1987).

7. Simmonds, S. H., and P. G. Strange. Inhibition of inositolphospholipid breakdown by D₂ dopamine receptors in dissociated bovineanterior pituitary cells. Neurosci. Lett. 60:267-272 (1985).

8. Enjalbert, A., F. Sladaczek, G. Guillon, P. Bertrand, C. Shu, J.Epelbaum, A. Garcia-Sainz, S. Jard, C. Lombard, C. Kordon, and J.Bockaert. Angiotensin II and dopamine modulate both cAMP and inositolphosphate productions in anterior pituitary cells: Involvement inprolactin secretion. J. Biol Chem. 261:4071-4075 (1986).

9. Judd, A. M., I. S. Login, K. Kovacs, P. C. Ross, B. L. Spangelo, W.D. Jarvis, and R. M. MacLeod. Characterization of the MMQ cell, aprolactin-secreting cloned cell line that is responsive to dopamine.Endocrinology 123:2341-2350 (1988).

10. Lefkowitz, R. J. and M. G. Caron. Adrenergic receptors: Models forthe study of receptors coupled to guanine nucleotide regulatoryproteins. J. Biol. Chem. 263:4993-4996 (1988).

11. Uhler, M. D., and G. S. McKnight. Expression of cDNAs for twoisoforms of the catalytic subunit of cAMP-dependent protein kinase. J.Biol. Chem. 262:15202-15207, 1987.

12. Gorman, C., R. Padmanabhan, and B. H. Howard. High efficiencyDNA-mediated transformation of primate cells. Science 231:551-553(1983).

13. Cieng, Y.-C. and W. H. Prusoff. Relationship between the inhibitionconstant (K_(I)) and the concentration of inhibitor which causes 50 percent inhibition (ISO) of an enzymatic reaction. Biochem. Pharmacol.22:3099-3108 (1973).

14. Salomon, Y., C. Londos, and M. Rodbell. A highly sensitive adenylatecyclase assay. Analyt. Biochem. 58:541-548 (1974).

15. Peterson, G. L. A simplification of the protein assay method ofLowry et al. which is more generally applicable. Analyt. Biochem.83:346-356 (1977).

16. Cote, T. E., E. A. Frey, C. W. Grewe, and J. W. Kebabian. Evidencethat the dopamine receptor in the intermediate lobe of the rat pituitarygland is associated with an inhibitory guanyl nucleotide component. J.Neural. Trans. Suppl. 18:139-147 (1983).

17. Hamblin, M. W., and I. Creese. ³H-Dopamine binding to rat seriatalD-2 and D-3 sites: Enhancement by magnesium and inhibition by guaninenucleotides and sodium. Life Sci. 30:1587-1595 (1982).

18. Weiss, S., M. Sebben, J. A. Garcia-Sainz, and J. Bockaert.D₂-Dopamine receptor-mediated inhibition of cyclic AMP formation instriatal neurons in primary culture. Mol. Pharmacol. 27:595-599 (1985).

19. Onali, P., M. C. Olianas, and G. L. Gessa. Characterization ofdopamine receptors mediating inhibition of adenylate cyclase activity inrat striatum. Mol. Pharmacol. 28:138-145 (1985).

20. Agui, T., N. Amlaiky, M. G. Caron, and J. W. Kebabian. Binding of[¹²⁵I]-N-(p-aminophenethyl)spiroperidol to the D-2 dopamine receptor inthe neurointermediate lob of the rat pituitary gland: A thermodynamicstudy. Mol. Pharmacol. 32:163-169 (1988).

21. Koch, S. W., B. K. Koe, and N. G. Bacopoulos. Differential effectsof the enantiomers of 3-(3-hydroxyphenyl)-N-n-propylpiperidine (3-PPP)at dopamine receptor sites. Eur. J. Pharmacol. 92:279-283 (1983).

22. Meller, E., K. Bohmaker, Y. Namba, A. J. Friedhoff, and M.Goldstein. Relationship between receptor occupancy and response atstriatal dopamine autoreceptors. Mol. Pharmacol. 31:592-598 (1987).

23. Borgundvaag, V., and S. R. George. Dopamine inhibition of anteriorpituitary adenylate cyclase is mediated through the high-affinity stateof the D₂ receptor. Life Sci. 37:379-386 (1985).

24. Abramson, S. N., M. W. Martin, A. R. Hughes, T. K. Harden, K. A.Neve, D. A. Barrett, and P. B. Molinoff. Interaction of β-adrenergicreceptors with the inhibitory guanine nucleotide-binding protein ofadenylate cyclase in membranes prepared from cyc-S49 lymphoma cells.Biochem. Pharmacol. 37:4289-4297 (1988).

25. Tashjian, A. H. Clonal strains of hormone-producing pituitary cells.Meth. Enzymol. 58:527-535 (1979).

26. Albert, P. R., K. Neve, J. Bunzow, and O. Civelli. Biologicalfunctions of the rat dopamine D₂ receptor cDNA expressed in GH₄C₁ ratpituitary cells. Proc. Endocrine Soc. 71: (in press).

Example 2

Summary

We have previously described a cDNA which encodes a binding site withthe pharmacology of the D₂-dopamine receptor (Bunzow, J. R., et al.(19868) Nature 336, 783-787). We demonstrate here that this protein is afunctional receptor, i.e., it couples to G-proteins to inhibit cAMPgeneration and hormone secretion. The cDNA was expressed in GH₄C₁ cells,a rat somatomammotrophic cell strain which lacks dopamine receptors.Stable transfectants were isolated and one clone, GH₄ZR₇, which had thehighest levels of D₂-dopamine receptor mRNA on Northern blot, wasstudied in detail. Binding of D₂-dopamine antagonist H-spiperone tomembranes isolated from GH₄ZR₇ cells was saturable, with K_(D)=96 pM,and B_(max)=2300 fmol/mg protein. Addition of GTP/NaCl increased theIC₅₀ value for dopamine competition for ³H-spiperone binding bytwo-fold, indicating that the D₂-dopamine receptor interacts with one ormore G proteins. To assess the function of the dopamine binding site,acute biological actions of dopamine were characterized in GH₄ZR₇ sells.Dopamine decreased resting intra- and extracellular cAMP levels by50-70% (EC₅₀=8±2 nM), and blocked completely VIP-induced enhancement ofcAMP levels (EC₅₀=6±1 nM), which ranged from 8-12 times basal levels.Antagonism of dopamine-induced inhibition of VIP-enhanced cAMP levels byspiperone, (+)-butaclamol, (−)-sulpiride and SCH23390 occurred atconcentrations expected from K_(I) values for these antagonists at theD₂-receptor and was stereo-selective. Dopamine (as well as severalD₂-selective agonists) inhibited forskolin-stimulated adenylate cyclaseactivity by 45±6%, with EC₅₀ of 500-800 nM in GH₄ZR₇ membranes.Dopaminergic inhibition of cellular cAMP levels and of adenylate cyclaseactivity in membrane preparations was abolished by pretreatment withpertussis toxin (50 ng/ml, 16 h). Dopamine (200 nM) abolished VIP andTRH-induced acute prolactin release. These data show conclusively thatthe cDNA clone encodes a functional dopamine-D₂ receptor which couplesto G proteins to inhibit adenylate cyclase, and both cAMP-dependent andcAMP-independent hormone secretion. The GH₄ZR₇ cells will prove usefulin elucidating further the biochemistry of the dopamine D₂ receptor.

Introduction

The major element controlling PRL¹ secretion from the pituitary is theconcentration of dopamine in the hypophyseal portal bloodstream (1).Dopamine acts via dopamine-D₂ receptors on pituitary lactotrophs toinhibit basal and hormone-stimulated secretion of PRL (1-5). Thedopamine-D₂ receptor interacts with pertussis toxin-sensitive,inhibitory G proteins (6-9) to reduce adenylate cyclase activity, and toblock enhancement of cAMP levels by other agents (6, 10-12). Dopaminealso decreases [Ca⁺⁺]_(i) in lactotrophs, and partially inhibitselevation of [Ca⁺⁺]_(i) by other agents, such as TRH (13-15). Bothdopaminergic inhibition of cAMP and of [Ca⁺⁺]_(i) are mediated throughcoupling to one or more pertussis toxin-sensitive G proteins, and appearto contribute to dopamine inhibition of PRL secretion (15). The preciserelation between these components of dopamine action has beeq difficultto study (15, 16) due to the presence of heterogeneous cell types,limitations of cell number, and variations in responsiveness of diverslactotroph preparations.

¹The abbreviations used are: PRL, prolactin; GH, growth hormone;[Ca⁺⁺]_(i), cytosolic free calcium concentration; VIP, vasoactiveintestinal peptide; TRH, thyrotropin-releasing hormone; IBMX,3-isobutyl-1-methyl xanthine; K_(D), equilibrium dissociation constant;EC₅₀ (IC₅₀), concentration required to elicit a half-maximal effect(inhibition).

The recent cloning of the dopamine-D₂ receptor cDNA (17) provides auseful tool to examine the intracellular actions and regulation of thereceptor. To examine whether the D₂-receptor clone directs synthesis ofa functional receptor, and to define the pathway between dopamine-D₂receptor activation and biological effect, we have transfected thedopamine-D₂ receptor cDNA into a pituitary-derived cell strain, GH₄C₁cells. GH₄C₁ cells are rat pituitary cells which synthesize and secretePRL and GH, and possess a variety of hormone, growth factor, andneurotransinitter receptors, second messenger systems, and ion channelsand have provided an accessible model of lactotroph function (18, 19).However, these cells lack dopamine-D₂ receptors, which are present onnormal lactotrophs, and thus provide an ideal host for studying thefunction of the dopamine-D₂ receptor. This report demonstrates that thegene product of the cDNA clone functions as a dopamine-D₂ receptor andcouples to inhibitory G proteins to decrease cAMP accumulation² and PRLrelease. The GH₄C₁ transfectants characterized herein should provide auseful cell system in which the mechanisms of dopamine action at D₂receptors may be studied further.

²These results have been presented in part at the 71^(st) AnnualEndocrine Meetings, Seattle, Wash., Abstract #1278.

Experimental Procedures

Materials: Dopamine agonists and antagonists were from ResearchBiochemicals Incorporated (Waltham, Mass.), except quinpirole (Lilly),bromocryptine (Sandoz Research Institute), and (+) and (−)3-PPP (Astra).Rabbit antibody (lot CA-3) to 2-O-succinyl-cAMP-bovine serum albuminconjugate was obtained from ICN (Irvine, Calif.), rPRL standard andanti-rPRL antibody were from Dr. Salvatore Raiti, NIDDK, Bethesda, Md.Peptides were from Peninsula (, Calif.) or Sigma (St. Louis, Mo.).α³²P-dCTP (2,200 Ci/mmol), ¹²⁵I-2-O-(iodotyrosyl methyl ester)-succinylcAMP (2,200 Ci/mmol), ¹²⁵I-rPRL (2,200 Ci/mmol), α³²P-ATP (10-50Ci/mmol), ³H-cAMP (31.9 Ci/mmol) were from New England Nuclear (Boston,Mass.). All other chemicals were reagent grade, obtained primarily fromSigma.

Methods:

Construction of pZEM-D₂-cDNA: The pZEM-3 plasmid (20) was cut at the BglII site between the metallothionein promotor and hGH 3′-flankingsequence. Full-length dopamine-D₂ cDNA (17) was excised from λGT10 withSal I and was ligated to the cut pZEM-3 plasmid in the presence of dATPand dGTP (250 μM) and transformed into E. Coli strain XL-1 (Stratagene).Recombinants were characterized by their hybridization to ³²P-labelledD₂-cDNA, followed by restriction analysis and DNA sequencing.Recombinants with cDNA inserts in the sense orientation were preparedand purified by CsCl gradient centrifugation for transfection intoeucaryotic cells.

Cell Culture: GH₄C₁ cells, obtained from Dr. A. H. Tashjian, Jr.(Harvard University, Boston, Mass.) and subdlones were grown in Ham'sF10 medium, supplemented with 10% fetal bovine serum, at 37° C. in 5%CO₂. For studies of ³H-spiperone binding or adenylate cyclase activity,cells were grown in Dulbecco's modified Eagle's medium supplemented with10% fetal bovine serum at 37° C. in 10% CO₂. Media were changed 12-24hprior to transfection or experimentation. For transfection, GH₄C₁ cellswere grown to 2-4×10⁶ cells/10 cm dish. 20 μg of pZEM-D₂cDNA and 1 μgpRSV-neo were co-precipitated with calcium phosphate in 2 ml ofHepes-buffered saline, and placed over cells for 10-20 min. 8 ml of warmF10+10% fetal calf serum (pH 7.0) was added, and the cells incubated for4-5 h, 37° C. The medium was removed and 15% glycerol in Hepes-bufferedsaline was added, and incubated for 3 min., 37° C. The plates wererinsed and fresh F10+10% fetal calf serum added, and the cells placed inthe incubator for 16-20 h. Fresh medium supplemented with 700 μg/ml G418(Geneticin, GIBCO, N.Y.) was added over the next 3-4 weeks, to selectfor stable transfectants expressing neo-resistance. Single colonies wereisolated using sterile micropipette tips to take up individual coloniesin 3-5 μl. Once stocks of the transfectant cell lines were stored frozenin liquid nitrogen, G418 was omitted from growth media.

RNA Isolation and Northern Blot Analysis: Cells were rinsed incalcium-free Hepes-buffered saline+0.02% EDTA and extracted withTris-buffered guanidinium hydrochloride, centrifuged (33,000 rpm, 16 h)through a 1.7 g/ml CsCl pad, and the pellets extracted withphenol/chloroform and ethanol precipitated (21). RNA was resuspended andquantitated by UV absorbance at OD=260 nm. For Northern blots, RNA wasdenatured in glyoxal/dimethylsulfoxide (1 h, 50° C.), and run on a 1%agarose gel in 10 mM sodium phosphate. RNA was blotted overnight ontonylon membrane (N-bond, Amersham), baked at 80° C. for 2 h.Prehybridization was as described, for 6 h at 42° C. Random-primed³P-labelled 1.6 kb BamHI-BglII fragment of the D₂-cDNA (1-2×10⁶ dpm/μg)was used for hybridization, 16-20 h at 42° C. in 50% formamide. Blotswere washed in 2×SSC for 10 min, room temperature, followed by 3×15 min.wash in 0.2×SSC, 0.5% SDS, 70° C., and exposed to X-ray film overnightat −80° C., with intensifying screen.

Ligand Binding: cell membranes were prepared by first replacing growthmedium with ice-cold hypotonic buffer (1 mM Hepes, pH 7.4, 2 mN EDTA).After swelling for 10-15 min, the cells were scraped from the plate andcentrifuged at 24,000 g for 20 min, lysed with a Brinkman Polytronhomogenizer at setting 6 for 10 sec in Tris-isosaline (50 mM Tris, pH7.4, 0.9% NaCl) and stored at −70° C. for receptor binding experiments,or resuspended in 50 mM Tris, pH 7.4, centrifuged as above, andresuspended in 50 mM Tris for immediate use in adenylate cyclase assay(below). For binding assays, the membrane preparation was thawed,centrifuged (24,000 g×20 min) and resuspended in Tris-isosaline exceptwhere indicated. Aliquots of membrane preparation were added to tubescontaining 50 mM Tris, pH 7.4, 0.9% NaCl, 0.025% ascorbic acid, 0.001%bovine serum albumin, ³H-spiperone and indicated drugs. (+)-Butaclamol(2 μM) was used to define nonspecific binding, which was less than 10%of total binding at concentrations of radioligand near the value. Assayswere carried out in duplicate, in a volume of 2 ml for saturationanalyses or 1 ml for inhibition analyses. Incubations were initiated byaddition of 10-50 μg of membrane protein, carried out at 37° C. for 50min, and stopped by addition of 10 ml of ice-cold buffer (10 mM Tris, pH7.4, 0.9% NaCl) to each tube. The samples were immediately filteredthrough glass-fibre filters (Schleicher and Schuell No. 30) and washedwith 10 ml of ice-cold buffer. Radioactivity retained on the filter wascounted using a Beckman LS 1701 scintillation counter. In experiments toexamine the effect of GTP on dopamine binding, cells were harvested,centrifuged, resuspended in Tris-Mg⁺⁺ (50 mM Tris, pH 7.4, 4 mM MgCl₂)and incubated for 15 min, 37° C. After centrifugation, the resuspendedprotein was added to assays containing Tris-Mg⁺⁺ with no added GTP orNaCl, or Tris-Mg++with 120 mM NaCl and 100 μm GTP.

cAMP and PRL Assay: Cells were plated in 6-well, 35 mm dishes, 3-7 daysprior to experimentation. Cells were pre-incubated in 2 ml/well warmF10+0.1% (−)-ascorbic acid+20 mM Tris (pH 7.2) (FAT) for 5-10 min,followed by addition of 1 ml/well of FAT+100 μm IBMX+experimentalcompounds, and incubated for 30 min. at 37° C. Experimental compoundswere diluted 200- to 1000-fold from stock solutions made immediatelyprior to assay. The final ethanol concentration never exceeded 0.1%, aconcentration without effect on basal or VIP-enhanced cAMP or PRL levelsin GH₄ZR₇ cells. Media were collected, and the cells were lysedimmediately in 1 ml of boiling water. Cell lysates and media werecentrifuged (2000×g, 10 min., 4° C.), and the supernatants collected forassay as cell extracts. Cell extracts and media samples were frozen at−20° C. until assay, if not assayed immediately. cAMP was assayed by aspecific radioimmunoassay as described (22), with antibody used at 1:500dilution. After 16 h incubation at 4° C., 20 μl of 10% BSA and 1 ml of95% ethanol were added consecutively to precipitate the antibody-antigencomplex. Standard curves showed IC₅₀ of 0.5±0.2 pmol using cAMP asstandard. PRL was assayed in the media samples obtained as describedabove, except that IBMX was omitted during the 30 min incubation in FATmedium. PRL levels were determined by specific radioimmunoassay usingStaphyloccus A lysate (IgSorb, The Enzyme Center, Malden, Mass.) toprecipitate antigen-antibody complexes (23). Standard curves gave IC₅₀of 36±6 ng using rPRL standard.

Adenylate Cyclase Assay: The conversion of α³²P-ATP to ³²P-cAMP wasdetermined essentially as described by Salomon et al. (24). Membranes(10-50 μg) were added in a volume of 100 μl to an assay of 200 μlcontaining 50 mM Tris, pH 7.4, 5 mM cAMP, 1 mM IBMX, 1 mM MgCl₂, 0.5 mMEGTA, 0.25 mM ATP, 30 μm GTP, and about 2×10⁶ cpm of α³²P-ATP, andvarious drugs. Assays carried out in triplicate were initiated bywarming to 25° C. and terminated by cooling to 0° C. Trichloroaceticacid (100 μl of 30% solution) was added to each assay, and ³H-cAMP(30,000 cpm) was added to each tube as an internal standard. The assayvolume was increased to 1 ml by addition of water, and tubes werecentrifuged (2000 g×10 min). cAMP in the supernatant was isolated bysequential chromatography on columns containing Dowex AG50W-X4 resin andneutral alumina. The 2-ml eluate from each alumina columntwas dissolvedin 10 ml of Bio-Safe II (RPI, Mount Prospect, Ill.) for liquidscintillation counting. Calculations: Data from cAMP and PRL assays areexpressed as means±standard error for triplicate determinations.Curve-fitting parameters were obtained by nonlinear regression analysisusing the Enzfitter program (Elsevier Biosoft). Average affinity, EC₅₀and IC₅₀ values are geometric means of the indicated number ofexperiments. In competition experiments, K_(I) values were calculatedfrom experimentally determined IC₅₀ values by the method of Cheng andPrusoff (25). All experiments were representative of 3-5 independenttrials, with the exception of that presented in FIG. 16 (2 trials).

Results

Characterization of Stable Transfectants: GH₄C₁ cells were cotransfectedwith pZEM-D₂-cDNA and pRSV-neo, and colonies resistant to the antibioticG418 were isolated and initially characterized by Northern blotanalysis. One clone, GH₄ZR₇, had higher levels of 2.5 kb D₂ mRNA thanother clones (FIG. 13A). Wild-type (untransfected) GH₄C₁ cells, as wellas a GH₄C₁ cell transfectant (GH₄ZD₁₀) expressing the rat 5-HT_(1A)receptor gene in the pZEM-3 vector³ showed no hybridization to theD₂-receptor probe. Pretreatment of GH₄ZR₇ cells with 100 μM ZnSO₄ for 36h induced a marked enhancement of D₂ receptor mRNA, indicating that thetranscribed mRNA is regulated by the zinc-sensitive metallothioneinpromotor (20). The GH₄ZR₇ clone was used for further analysis below)because of the high levels of dopamine-D₂ receptor expression in thisclone.

³Albert, P. R., et al., manuscript in preparation.

Specific binding of the selective dopamine-D₂ receptor antagonist,³H-spiperone, was assayed in crude membranes prepared from GH₄ZR₇ cells(FIG. 13B). The GH₄ZR₇ membranes showed a saturable component of³H-spiperone binding which was displaced by 2 μM (+)-butaclamol, whereasmembranes from wild-type GH₄C₁ cells showed no specific ³H-spiperonebinding (data not shown). In 5 experiments, the GH₄ZR₇ cell membranesshored maximal specific ³H-spiperone binding of 2046±315 fmol/mg ofprotein, and a mean 4 value of 96±1 pM. These values demonstrate robustexpression of dopamine-D₂ binding sites receptors in these cells withaffinity for ³H-spiperone comparable to that obtained in rat striatalmembranes, and in Ltk⁻ cells transfected with pZEM-D₂-cDNA (17).

To ascertain whether the expressed dopamine binding site interacted witha G protein, inhibition of ³H-spiperone binding by dopamine was assayedin GH₄ZR₇ cell membranes, in the absence or presence of 100 μM GTP and120 mM NaCl (FIG. 13C). Assays were carried out in the presence of 4 mMMgCl₂ to promote high affinity binding of dopamine. In the absence ofadded GTP and NaCl, dopamine inhibited ³H-spiperone binding withIC₅₀=49±15 μM, and Hill coefficient of 0.69, suggesting the presence ofhigh and low affinity sites for dopamine. Analyzing the data accordingto a model assuming the presence of two classes of binding sitesindicated that 46±15% of the receptors had a high affinity (K_(D)=0.5μM) for dopamine and the remaining receptors had lower affinity (30 μM)for the agonist. In the presence of GTP and NaCl, the IC₅₀ for dopaminewas shifted two-fold to 109±20 μM (K_(I)=17 μM) with Hill coefficientcloser to unity (0.93). Thus, the presence of GTP/NaCl converts theidopamine receptors from a heterogeneous population of high and lowaffinity receptors to a nearly homogeneous population of receptors in alow-affinity agonist state, as observed in striatal membranepreparations (6, 7). These data suggest that the cloned dopamine bindingsite interacts with G proteins when expressed in GH₄ cells.

Dopamine Actions on cAMP and PRL Levels: To test directly the functionof the expressed dopamine-D₂ receptor clone, the actions of dopamine oncellular cAMP levels were measured. These assays were conducted in thepresence of 100 μM IBMX, to inhibit phosphodiesterase activity in thesecells (22). Thus, the observed changes in cAMP levels reflect changes inthe rate of synthesis of cAMP rather than changes in its degradation.Dopamine actions on basal cAMP levels were measured, as well as dopamineinhibition of VIP-enhanced levels of cAMP. GH₄C₁ cells respond to VIPwith an enhancement of cAMP accumulation (FIG. 14A) as described byothers (22, 26). Dopamine had no effect on extracellular cAMP levels inwild-type GH₄C₁ cells, whether VIP was omitted or present during theincubation. This result is consistent with the lack of D₂-dopaminereceptor mRNA and binding in GH₄C₁ cells (FIGS. 13A through 13C), andindicates that these cells also lack a detectable D₂-dopamine responsesince dopamine does not elevate cAMP concentrations. In media fromGH₄ZR₇ cells, dopamine inhibited both basal cAMP levels (by 50-70%), andreduced VIP-enhanced cAMP to basal levels. These actions of dopamine toreduce cAMP levels were consistently observed in all experiments anddopamine was equally effective in lowering intracellular cAMP levels(FIG. 14B). As observed previously in GH₄C₁ cells (22), both intra- andextracellular cAMP levels change in parallel, although changes inextracellular cAMP may be more pronounced due to lower recovery ofextracted intracellular cAMP. Dopamine actions on cAMP accumulation wereblocked by (−)-sulpiride, a highly selective dopamine-D₂ antagonist,whereas the inactive stereoisomer, (+)-sulpiride, did not blockdopaminergic inhibition of cAMP accumulation. Stereo-selective blockadeby sulpiride suggested that inhibition of cAMP levels in GH₄ZR₇ bydopamine was mediated by activation of a dopamine-D₂ receptor notpresent in wild-type GH₄C₁ cells.

The physiological outcome of dopamine action is inhibition of secretion,which was assayed by measuring acute (30 min) PRL release in GH₄ZR₇cells (FIG. 14D). VIP and TRH enhanced PRL secretion 1.5- and 3-fold,respectively. VIP is thought to enhance PRL release by a cAMP-dependentmechanism (22, 23, 26), while TRH acts by a cAMP-independent mechanismlinked to calcium mobilization (19, 27). Dopamine did not inhibit basalPRL release, but both VIP- and TRH-induced enhancement of PRL secretionwere blocked by dopamine. Thus, dopamine blocked both cAMP-dependent andcAMP-independent secretion in GH₄ZR₇ cells. These actions of dopaminewere reversed by (−)-sulpiride, but not by (+)-sulpiride. In GH₄C₁cells, dopamine had no effect on basal, VIP-stimulated, orTRH-stimulated secretion of PRL (data not shown).

To examine whether concentrations required for biological responsecorrelated with affinity for the dopamine-D₂ receptor, dose-responserelations were examined for dopamine actions on cAMP levels (FIGS. 15Aand 15B). Dopamine potently inhibited intra- and extracellular levels ofcAMP with similar EC₅₀ values. Furthermore, dopamine inhibited bothbasal and VIP-enhanced cAMP accumulation with EC₅₀ values of 8±2 nM and6±1 nM, respectively. These data demonstrate that dopamine inhibits bothbasal and stimulated cAMP accumulation with approximately equal potency.The high potency of these inhibitory actions of dopamine supports theassertion that GH₄ZR₇ cells express a functional dopamine-D₂ receptor.

The pharmacological specificity of dopaminergic inhibition ofVIP-enhanced levels of cAMP in GH₄ZR₇ was examined further usingspecific receptor antagonists (FIG. 16). The data show that certainreceptor antagonists reverse dopamine-induced inhibition of VIP-enhancedlevels of extracellular cAMP. Maximal cAMP (100%) corresponded to cAMPlevels in the presence of VIP alone. Low concentrations of dopamine-D₂antagonists (spiperone, (+)-butaclamol, (−)-sulpiride) blocked dopamineaction, whereas SCH23390, a specific dopamine-D₁ antagonist, was activeonly at very high concentrations. Inactive stereoisomers ofD₂-antagonists ((−)-butaclamol, (+)-sulpiride (FIG. 14C)) had little orno effect on dopamine action. Antagonists added in the absence ofdopamine did not alter cAMP concentrations. Estimated K_(I) valuesobtained from IC₅₀ values for the antagonists (see description of FIG.16) were similar to values determined from binding competition studiesof the dopamine D₂ receptor (17), showing that inhibition of cAMP levelsby dopamine in GH₄ZR₇ cells is mediated by a receptor which ispharmacologically indistinguishable from the dopamine-D₂ receptor.

Inhibition of Adenylate Cyclase: To assess directly inhibition ofadenylate cyclase activity by dopamine receptor agonists, the conversionof ³²P-ATP to ³²P-cAMP was measured in membranes prepared from GH₄ZR₇cells (FIGS. 17A and 17B). Dopamine inhibited total forskolin (10μM)-stimulated activity by 45% with an average EC₅₀ value of 0.36 μM(n-5). As observed in pituitary (11) and striatal (28) membranes,bromocryptine behaved as partial agonist, maximally inhibiting enzymeactivity by 23% (EC₅₀=6 nM). Inhibition of adenylate cyclase activity byselective D₂-agonists was stereo-selective. For example, quinpiroleinhibited forskolin-stimulated cyclase activity by 41% (EC₅₀=0.32 nM),whereas LY181990, the inactive (+)-enantiomer of quinpirole, cause noconsistent reduction in enzyme activity. Similarly, (+)-3-PPP (EC₅₀=0.86nM) was as efficacious as dopamine, whereas the enantiomer (−)-3-PPP didnot consistently reduce adenylate cyclase activity. VIP also stimulatedadenylate cyclase activity in GH₄ZR₇ cell membranes, as reported forwild-type GH₄C₁ cell membranes (29). Total activity stimulated by 200 nMVIP was 22±7 pmol/mg protein/min (n=3), and VIP-enhanced activity wasinhibited 41% by dopamine (100 μM), compared to 50-55% inhibition in ratanterior pituitary membranes (11). No effect of dopamine on basaladenylate cyclase activity was observed in these preparations.Nevertheless, inhibition by dopamine of forskolin- or VIP-stimulatedadenylate cyclase activity provides a likely mechanism for inhibition ofcAMP accumulation by dopamine in GH₄ZR₇ cells.

Pertussis Toxin Sensitivity: Sensitivity to pertussis toxin is ahallmark of receptors, such as the dopamine-D₂ receptor (6-12, 15),which couple to inhibitor G proteins (e.g., G_(i) or G_(o)) to induceresponses. Pretreatment of GH₄ZR₇ cells with pertussis toxin for 16 h(FIGS. 12A through 12C) uncoupled dopamine-mediated inhibition offorskolin-stimulated membrane adenylate cyclase activity, and abolishedinhibition of basal and VIP-stimulated cAMP accumulation by dopamine.The concentration of pertussis toxin and incubation time used producemaximal blockage of somatostatin responses in wild-type cells (30), andthe dopamine responses were almost completely inhibited under theseconditions. By contrast, basal and VIP-stimulated cAMP accumulation, aswell as basal and forskolin-stimulated cyclase activity, were notsignificantly altered by pertussis toxin pretreatment. These datasupport the assertion that the expressed cDNA clone codes for adopamine-D₂ binding site which is functionally coupled to inhibitory Gproteins present in GH₄ cells, and thus represents a bona fide receptor.

Discussion

The cDNA clone coding for a dopamine-D₂ binding site (17) was expressedin GH₄C₁ cells to determine whether the clone expresses a functional D₂receptor, which is coupled by pertussis toxin-sensitive inhibitory Gproteins to inhibition of adenylate cyclase, cAMP accumulation, andinhibition of PRL secretion (6-12, 15). Dopamine-D₂ receptors wereexpressed specifically from a D₂-cDNA construct under the regulation ofthe mouse metallothionein promotor (20), as evidenced by the presence ofD₂ receptor mRNA in the GH₄ZR₇ transfectant. Dopamine-D₂ receptor mRNAlevels were undetectable in untransfected GH₄C₁ cells, or in cellstransfected with the rat 5-HT1A receptor subtype (FIG. 13A). The mRNAspecies found in GH₄ZR₇ cells was approximately the same molecularweight as D₂ receptor mRNA found in rat brain (17). Levels ofdopamine-D₂ mRNA increased by addition of 100 μM Zn⁺⁺ in GH₄ZR₇ cells,indicating that expression of the mRNA is controlled by theZn⁺⁺-sensitive mouse metallothionein promotor, and does not representtranscription of the endogenous gene. Specific binding of ³H-spiperonebinding was present only in GH₄ZR₇ cells, and was increased by 100 μMZn⁺⁺, and thus correlated with the expression of dopamine-D₂ receptormRNA in these cells. The D₂-receptor mRNA was transcribed to yieldrobust expression of high affinity dopamine-D₂ binding sites in GH₄ZR₇cells.

The presence of dopamine-D₂ binding in the GH₄ZR₇ transfectantcorrelated with potent and powerful inhibition of cAMP accumulation andPRL release, as well as inhibition of forskolin-stimulated adenylatecyclase activity, actions of dopamine not observed in untransfectedGH₄C₁ cells. These inhibitory actions of dopamine match exactly theknown physiological actions of dopamine in pituitary lactotrophs (1, 2).In particular, dopamine controls PRL secretion and cAMP accumulation inlactotrophs such that stimulation of these processes does not occurunless dopamine concentrations decrease to low levels (1, 3-5).Similarly, in the present of maximal concentrations of dopamine, VIPdoes not enhance cAMP levels or PRL secretion in GH₄ZR₇ cells (FIGS. 14Athrough 14C). The potency of dopamine inhibition of basal andVIP-enhanced cAMP accumulation in GH₄ZR₇ cells (FIGS. 15A through 15B)was in the range of concentration expected for lactotrophs, given thatdopamine concentrations in hypophyseal portal blood vary from 7 nM infemale rates during proestrous, to 20 nM during estrous, and are 3 nM inmale rats (31). Detailed analysis of the pharmacology ofdopamine-induced inhibition of adenylate cyclase and cAMP accumulationusing specific agonists and antagonists are fully consistent with theconclusion that dopaminergic actions in GH₄ZR₇ cells are mediated by areceptor indistinguishable from the dopamine-D₂ receptor.

The discrepancy between the measured affinity of dopamine (FIG. 13C),and the potency of dopamine to inhibit cAMP accumulation (FIGS. 13Athrough 13C) raises the possibility that GH₄ZR₇ cells have “spare”receptors, i.e., a sufficient excess of binding sites to shift the EC₅₀for biological action to values lower than the K_(D) value. Analternative explanation is that the receptor in membrane preparationshas a lower affinity for agonists (but unchanged affinity forantagonists since measured IC₅₀ values correlated with K_(I) values)than in intact cells. Since cytosolic or membrane-associated componentspresent in intact cells are not entirely replaced in membrane bindingand adenylate cyclase assay conditions, it is possible that componentswhich allow for optimal function of the dopamine receptor in membranepreparations are lacking. This assertion is supported by the EC₅₀ valuefor inhibition of particulate adenylate cyclase by dopamine (360 nM),which is close to K_(I) values obtained for dopamine from bindingcompetition experiments (500 nM), but 100-fold higher EC₅₀ values (6-8nM) obtained for inhibition of cAMP levels by dopamine in intact cells.This difference in conditions may explain the observed differencesbetween assays in intact versus particulate preparations. However,affinities of Antagonists correlated well with estimated K_(I) valuesobtained from cAMP accumulation experiments (FIG. 16), indicating thatantagonist binding is similar in membranes and whole cells.

Further evidence of coupling of the expressed dopamine-D₂ binding siteto G proteins is the shift of dopamine binding affinity to loweraffinity in the presence of GTP. Such GTP-induced shifts in affinityhave been reported for dopamine binding in membranes from rat brain (6,7), and are due to interaction of the receptor with G proteins (8, 9).In the presence of GTP, the G protein dissociates from the receptorleaving the receptor in a low affinity agonist state (6, 7). In the caseof dopamine, the difference between the affinities of the two states issmall, hence the GTP-induced shift to the low-affinity state is small(two-fold) and requires the presence of Na⁺ ion to maximize dissociationof the G protein (28). The observation of a GTP-induced shift indopamine affinity suggests that the expressed dopamine receptor isassociated with one or more G proteins in GH₄ZR₇ cell membranes.

Although the inhibitory actions of dopamine indicate that the receptorcouples to inhibitory G proteins, this was tested more directly bypretreating GH₄ZR₇ cells with pertussis toxin to inactivate inhibitory Gproteins (30). Pertussis toxin pretreatment completely blocked dopamineactions, without altering basal or VIP-stimulated cAMP accumulation.Thus, pertussis toxin prevented the dopamine-enhanced transduction ofbiological responses, presumably by uncoupling the dopamine-D₂ receptorfrom inhibitory G proteins. Unlike other systems (32), but as seen inwild-type GH₄C₁ cells (30), enhancement of cAMP levels by stimulators(e.g., VIP) was not augmented in pertussis toxin-treated GH₄ZR₇ cells,nor were basal cAMP levels altered by pertussis toxin. This suggeststhat inhibitory G proteins present in GH₄ZR₇ cells do not inhibit cAMPgeneration tonically.

The presence of somatostatin receptors on GH₄C₁ cells (33) allows fordirect comparison of the inhibitory actions of somatostatin anddopamine. Like dopamine, somatostatin does not inhibit basal adenylatecyclase activity (29) or basal PRL secretion (30). Somatostatin-inducedinhibition of VIP-stimulated cyclase activity (29), VIP-enhanced cAMPaccumulation (22), and PRL secretion (23, 30) in GH₄C₁ cells wereall-about half the maximal inhibition induced by dopamine in GH₄ZR₇cells. Indeed, in GH₄ZR₇ cells, somatostatin was half as effective asdopamine at inhibiting VIP-enhanced cAMP accumulation (data not shown).The larger effects of dopamine were not due to the presence of anexcessive number of D₂ receptors since the receptor number underconditions of cAMP accumulation and PRL secretion experiments was lessthan 10,000 sites/cell, smaller than somatostatin receptor number(13,000 sites/cell) in GH₄C₁ cells (33). It is apparent that thedopamine receptors expressed in these cells are more effective attransducing inhibitory actions than somatostatin receptors, suggesting amore effective coupling of the dopamine receptor to the G proteinspresent in GH cells.

A second reason for expressing the dopamine-D₂ receptor cDNA in GH₄C₁cells was to establish a new model system in which to study dopamineactions and D₂ receptor mechanisms. Current studies on dopamine actionin rat lactotrophs, the most accessible cell system expressingdopamine-D₂ receptors until recently (16), have come to divergentconclusions on mechanisms of dopamine inhibition. In neurons, dopaminehyperpolarizes membrane potential (34) by opening potassium channels(35), actions mediated by coupling of the D₂ receptor to inhibitory Gproteins (15). Membrane hyperpolarization induced by dopamine inlactotrophs (36) would close calcium channels, decreasing basal calciuminflux and explaining observed decreases in basal [Ca⁺⁺]_(i) induced bydopamine (13-15, 37, 38). However, dopamine has been observed toincrease [Ca⁺⁺]_(i) in certain pituitary cells (37). By examiningdopamine-induced changes in [Ca⁺⁺]_(i) in GH₄ZR₇ cells it will bepossible to specifically associate the D₂ receptor with changes in[Ca⁺⁺]_(i), and to ascertain the role of [Ca⁺⁺]_(i) in mediatingdopamine inhibition of hormone secretion. Another unresolved issue isthe mechanism by which dopamine inhibits enhancement of secretion and bycalcium-mobilizing hormones such as TRH. While some report inhibition bydopamine of TRH-induced enhancement of phosphatidyl inositol turnover(39, 40) and [Ca⁺⁺]_(i) (13, 14), others find no change (38, 41). Theobserved inhibition by dopamine of TRH-induced PRL release in GH₄ZR₇cells (FIG. 14C) suggests that dopamine may be coupled by G proteins toprocesses (e.g., opening of potassium channels) which alter TRH-inducedcalcium-mobilization or phosphatidyl inositol turnover. GH₄ZR₇ cellswill provide a homogeneous, abundant, and highly-responsive preparationin which to study these and other questions regarding mechanisms of thedopamine-D₂ receptor.

The data presented in this report indicate that the expressed clone (17)possesses the pharmacology of the dopamine-D₂ receptor in all actionsinvestigated including inhibition of PRL secretion, cAMP generation, andadenylate cyclase activity. The D₂ clone meets five basic criteria forclassification as a functional G protein-coupled receptor: 1) the cDNAclone for the dopamine-D₂ binding site possesses the archetypicalstructure of G protein-coupled receptors; 2) the clone expresses aprotein with saturable and specific binding properties; 3) agonistbinding affinity to the expressed binding site is decreased in thepresence of GTP; 4) the expressed receptor is coupled to functions(e.g., inhibition of adenylate cyclase) known to be regulated by Gproteins; 5) agents (e.g., pertussis toxin) which uncouple G proteinfunction uncouple activation of the expressed receptor from generationof the appropriate response. In conclusion, the interaction of thecloned dopamine-D₂ receptor with G proteins is productive, leading toactivation of α_(i) or α_(o) subunits and consequent inhibition of cAMPand PRL levels by both cAMP-dependent and cAMP-independent mechanisms.

References for Example 2

1. Ben-Jonathan, N. (1985), Endocr. Rev. 6, 564-589.

2. Memo, M., Missale, C., Carruba, M. O., and Spane, P. F. (1986), J.Neural Transm. (Suppl.) 22, 19-32.

3. Martinez de la Escalera, G., and Weiner, R. I. (1986), Endocrinology123, 1682-1687.

4. Lopez, F. J., Dominguez, J. R., Sanchez-Criado, J. E., andNegro-Vilar, A. (1988), Endocrinology 124, 527-535.

5. Ibid, pp. 536-542.

6. Sibley, D. R., DeLean, A., and Creese, I. (1982), J. Biol. Chem. 257,6351-6361.

7. DeLean, A., Kilpatrick, B. F., and Caron, M. G. (1982), Mol.Pharmacol. 22, 290-297.

8. Senogles, S. E., Benovic, J. L., Amlaiky, N., Unson, C., Milligan,G., Vinitsky, R., Spiegel, A. M., Caron, M. G. (1987), J. Biol. Chem.262, 4860-4867.

9. Ohara, K., Haga, K., Berstein, G., Haga, T., Ichiyama, A., and Ohara,K. (1988), Mol. Pharmacol. 33, 290-296.

10. DeCamilli, P., Macconi, D., and Spada, A. (1979), Nature 278,252-254.

11. Onali, P., Schwartz, J. P., and Costa, E. (1981), P.N.A.S. 78,6531-6534.

12. Cronin, M. J., Myer, G. A., MacLeod, R. M., and Hewlett, E. (1983),Am. J. Physiol. 244, E499-E504.

13. Schofield, J. G. (1983), F.E.B.S. Lett. 159, 79-82.

14. Margaroli, A., Vallar, L., Elahi, F. R., Pozzan, T., Spada, A., andMeldolesi, J. (1987), J. Biol. Chem. 261, 13920-13927.

15. Vallar, L. and Meldolesi, J. (1989), TIPS 10, 74-77.

16. Judd, A. M., Login, I. S., Kovacs, K., Ross, P. C., Spangelo, B. L.,Jarvis, W. D., and MacLeod, R. M. (1988), Endocrinology 123, 2341-2350.

17. Bunzow, J. R., VanTol, H. H. M., Grandy, D. K., Albert, P., Salon,J., Christie, M., Machida, C., Neva, K. A., and Civelli, O. (1988),Nature 336, 783-787.

18. Tashjian, A. H., Jr. (1979), Meth. Enzymol. 58, 526-535.

19. Ozawa, S., and Sand, O. (1986), Physiol. Rev. 66, 887-952.

20. Uhler, M., and McKnight, G. S. (1987), J. Biol. Chem. 262,15202-15207.

21. Chirgwin, J. M., Prybyla, A. E., MacDonald, R. J., and Rutter, W. J.(1979).

22. Dorflinger, L. J., and Schonbrunn, A. (1983), Endocrinology 113,1541-1550.

23. Dorflinger, L. J., and Schonbrunn, A. (1983), Endocrinology 113,1551-1558.

24. Salomon, Y., Londos, C., and Rodbell, M. (1974), Analyt. Biochem.58, 541-548.

25. Cheng, Y.-C., and Prusoff, W. H. (1973), Biochem. Pharmacol. 22,3099-3108.

26. Gourdji, D., Bataille, D., Vauclin, N., Grouselle, D., Rosselin, G.,and Tixier-Vidal, A. (1979), FEBS Lett. 104, 165-168.

27. Albert, P. R., and Tashjian, A. H., Jr. (1984), J. Biol. Chem. 259,15350-15363.

28. Onali, P., Olianas, M. C., and Gessa, G. L. (1985), Mol. Pharmacol.28, 138-145.

29. Koch, B. D., and Schonbrunn, A. (1984), Endocrinology 114,1784-1790.

30. Koch, B. D., Dorflinger, L. D., and Schonbrunn, A. (1985), J. Biol.Chem. 260, 13138-13145.

31. Ben-Jonathan, N., Oliver, C., Weiner, H. J., Mical, R. S., andPorter, J. C. (1977), Endocrinology 100, 452-458.

32. Kurose, H., Katada, T., Amano, T., and Ui, M. (1983), J. Biol. Chem.258, 4870-4875.

33. Schonbrunn, A., and Tashjian, A. H., Jr. (1978), J. Biol. Chem. 253,6473-6483.

34. Bunney, B. S., Aghajanian, G. K., and Roth, R. H. (1973), Nature(New Biol.) 245, 123-125.

35. Lacey, M. G., Mercuri, N. B., and North, R. A. (1987), J. Physiol.392, 397-416.

36. Taraskevich, P. S., and Douglas, W. W. (1978), Nature 276, 832-834.

37. Winiger, B. P., Wuarin, F., Zahan, G. R., Wollheim, C. B., andSchlegel, W. (1987), Endocrinology 121, 2222-2228.

38. Law, G. J., Pachter, J. A., and Dannies, P. (1988), Mol. Endocrinol.2, 966-972.

39. Journot, L., Homburger, V., Pantaloni, C., Priam, M., Bockaert, J.,and Enjalbert, A. (1987), J. Biol. Chem. 262, 15106-15110.

40. Vallar, L., Vicentini, L. M., and Meldolesi, J. (1988), J. Biol.Chem. 263, 10127-10134.

41. Canonico, P. L., Jarvis, W. D., Judd, A. M., and MacLeod, R. M.(1986), J. Endocrinol. 110, 389-393.

Example 3

Summary

A clone encoding a human dopamine D₂ receptor was isolated from apituitary cDNA library and sequenced. The deduced protein sequence is96% identical with that of the cloned rat receptor [Bunzow et al. (1988)Nature 336, 783-787] with one major difference: The human receptorcontains an additional 29 amino acids in its putative third cytoplasmicloop. Southern blotting demonstrated the presence of only one humandopamine D₂ receptor gene. Two overlapping phage containing the genewere isolated and characterized. DNA sequence analysis of these clonesshowed that the coding sequence is interrupted by six introns and thatthe additional amino acids present in the human pituitary receptor areencoded by a single exon of 87-basepairs. The involvement of thissequence in alternative splicing and its biological significance arediscussed.

Introduction

Dopamine neurons in the vertebrate central nervous system are involvedin the initiation and execution of movement, the maintenance ofemotional stability, and the regulation of pituitary function. Severalhuman neurological diseases, including Parkinson's Disease (1) andschizophrenia (2), are thought to be manifestations of imbalancesbetween dopamine receptors and dopamine. The receptors which mediatedopamine's effects have been divided into D₁ and D₂ subtypes, which aredistinguished by their G-protein coupling (3, 4), ligand specificities,anatomical distribution and physiological effects (5). The dopamine D₂receptors have been of particular clinical interest due to theirregulation of prolactin secretion (6) and their affinity forantipsychotic drugs (7, 8).

The dopamine D₂ receptors belong to the family of G-protein coupledreceptors. The sequence similarity shared by members of this familyenabled us to clone a rat brain dopamine D₂ receptor cDNA (9). We haveused that clone to isolate the human pituitary dopamine D₂ receptor cDNAdescribed here. We have found that the deduced amino acid sequences ofthese two receptors are very similar, with one notable difference. Thehuman receptor contains an additional 29 amino acids in its putativethird cytoplasmic loop which are encoded by one of the gene's exons.This genomic organization suggests that the existence of two dopamine D₂receptor mRNAs is the result of an alternative splicing event.

Materials and Methods

Cloning of the Human Pituitary cDNA

Human pituitary tissue was a generous gift from Drs. N. Seidah and M.Chretien, Clinical Research Institute of Montreal, Canada. Poly(A)⁺ mRNAand cDNA were prepared as previously described (9). The cDNA wassize-selected (1-6 kb) on agarose gels, isolated using Geneclean (Bio101), ligated to EcoRI adaptors, cloned into λGT10 arms (stratagene),and packaged (Gigapak Gold). Recombinants (1.5×10⁶) were screened onreplica nylon filters (DuPont Plaque/Colony Hybridization filters) with[³²P]-labelled hybridization probes. Prehybridization and hybridizationwere performed in 50% formamide, 1% SDS, 2×SSC (1×SSC=0.15 M NaCl/0.015M sodium citrate, pH 7) at 37° C. The complete sequence of both strandsof DNA were determined in M13mp19 using Sequenase (U.S. Biochemical)primed with synthetic oligonucleotides.

Expression and Pharmacology

The 2.5-kb human pituitary cDNA (hPitD₂) was cloned into pZem3 (a giftfrom Dr. E. Mulvihill, Zymogenetics) and co-transfected with the pRSVneogene into mouse Ltk⁻ cells by CaPo₄ precipitation (10). A stabletransfectant (L-hPitD₂Zem) was selected and maintained in 750 μg/ml ofG418 (Geneticin sulphate, Gibco). Twenty hours prior to the harvestingof membranes, these cells were incubated with 70 μM zinc sulphate.Membranes were prepared from L-HPitD₂Zem, from the Ltk⁻ cell lineexpressing the cloned rat dopamine D₂ receptor (L-RGB2Zem-1) and fromfreshly-dissected rat striata (Taconic Farm, Germantown, N.Y.), aspreviously described (11, 12). For the binding assays, membrane proteinwas used at 10-15 μg from L-hPitD₂Zem, 50-75 μg from L-RGB2Zem-1, and220-250 μg from rat striatum. The binding assays were incubated at 37°C. for 60 minutes in 50 mM Tris-HCl, 120 mM NaCl, 5 mM KCl, 2 mM CaCl₂,1 mM MgCl₂, pH 7.4, and then stopped by rapid filtration over glassfiber filters (Schleicher and Schuell, No. 32) which had been presoakedin 0.5% polyethyleneimine. The filters were washed twice in ice cold 50mM Tris-HCl, pH 7.4. Saturation curves were generated using increasingconcentrations of the highly D₂-specific antagonist [³H]-domperidone(13). Antagonist drugs were evaluated for their ability to inhibitspecifically bound [³H]-domperidone (1 nM). The B_(max), K_(d), and IC₅₀values were determined as previously described (14).

Southern Blotting

Human genomic DNA prepared from a normal male donor was a gift from M.Litt. Three micrograms of DNA were digested with restriction enzymes andthe fragments were electrophoresed in 0.7% agarose and blotted ontonitrocellulose filters (Schleicher and Schuell). Prehybridization andhybridization were performed at 37° C. in 50% formamide as previouslydescribed (15).

Genomic Sequencing

Genomic bacteriophage lambda libraries, prepared from normal human maleDNA, were purchased from Stratagene and Clontech Laboratories, Inc. andscreened with portions of the cloned rat dopamine D₂ receptor cDNA. DNAsequence was determined by a genomic sequencing approach (16, 17).Briefly, for each restriction enzyme used, cloned genomic phage DNA (50μg) was digested, subjected to chemical cleavage, and the resultingfragments resolved in a denaturing polyacrylamide gel. The DNA was thentransferred from the gel and immobilized onto nylon filters (PlascoGenetran). Using [³²P] end-labelled synthetic oligomers, ladders ofsequence were visualized within exons and read into neighboring introns.The filters were then either reprobed with a different oligomer, or anew filter was made in order to read the complementary sequence backacross the exon. Both strands of the coding region were sequenced.

Results

Cloning and Sequence Analysis of the Human Pituitary cDNA

Using the rat brain D₂ receptor cDNA as probe, three partial cDNAs wereisolated from a human pituitary library and sequenced. Twooligonucleotide probes based on these sequences were used to isolate afourth cDNA, hPitD₂, which encoded a full-length receptor protein (FIGS.18A through 18J). The human pituitary receptor contains seven putativetransmembrane domains and lacks a signal sequence. Overall, the humanand rat nucleotide sequences are 90% similar and show 96% identity atthe amino acid level. Several consensus sequences for N-linkedglycosylation, protein kinase A phosphorylation and palmitoylation (18)are conserved between the human and rat receptors. There are also 18amino acid differences (including one deletion) between these proteins,and, strikingly, the human pituitary receptor contains an additional 29amino acids in its putative third cytoplasmic loop.

Expression and Pharmacological Evaluation of hpit D₂ cDNA

In order to evaluate the pharmacological characteristics of the humanpituitary receptor, its cDNA was subcloned into pZem3 and expressed inmouse Ltk⁻ cells (L-hPitD₂Zem). Membranes prepared from these cellsshowed specific binding of [³H]-domperidone, a D₂-selective antagonist(12, 13), with a B_(max) of 4.05+/−0.3 pmol per mg (n=2) protein and aK_(d) of 0.74+/−0.11 nM (n=2). This K_(d) value is in excellentagreement with the published value of 0.74 nM in mouse brain membranes(13). A Scatchard plot of the data was linear. There was no detectable[³H]-domperidone binding in membranes prepared from cells transfectedwith pZem3 alone (data not shown). [³H]-domperidone binding toL-hPitD₂Zem membranes was inhibited by a number of dopamine D₂-specificdrugs (FIG. 19). Their rank order of potency was: spiperone,(+)-butaclamol, haloperidol, and sulpiride. The serotonin-selectiveantagonist mianserin and the D₁-selective antagonist SCH-23390 inhibiteddomperidone binding only at very high concentrations, as did theinactive isomer (−)-butaclamol. These values are essentially identicalto those obtained with membranes from Ltk⁻ cells transfected with thecloned rat dopamine D₂ receptor cDNA (L-RGB2Zem-1) and from rat striatum(FIG. 22).

Human Dopamine D₂ Receptor Gene

Using portions of the rat cDNA as probe, a clone was isolated from ahuman genomic library. This genomic clone, λHD2G1, contained a 1.6-kbBamHI fragment which encoded the last 64 amino acids of the human D₂receptor and 1.2-kb of 3′ non-coding sequence. The 1.6-kb fragment wasused to probe a Southern blot of human genomic DNA digested with threerestriction enzymes. Each enzyme generated a single fragment thathybridized to the probe (FIG. 20), indicating that there is probablyonly one human dopamine D₂ receptor gene.

In order to isolate a genomic clone that encoded the N-terminus of thehuman receptor protein, a 118-bp restriction fragment from the clonedrat dopamine D₂ receptor cDNA (corresponding to amino acid residues1-39) was used to screen a second genomic library. λHD2G2 was isolatedand found to overlap with λHD2G1 by 400 nucleotides (FIG. 21, line a).Together, these phage span 34-kb of the human dopamine D₂ receptor genelocus, DRD2 (19), and contain the sequence found in the hPitD₂ cDNA plussequences that extend 15-kb downstream of the polyadenylation signal and3.7-kb upstream of the translation initiation site. To characterize theintron/exon structure of the gene, a genomic sequencing approachemploying oligonucleotide probes and chemical cleavage was used (FIG. 21line b). Since the divergence of nucleotide sequences between human andrat members of this receptor family is approximately 10% (unpublishedobservations), we were able to initiate the genomic sequencing relyingon hybridization probes and restriction sites that are present in thecloned rat dopamine D₂ receptor cDNA. Our results demonstrate that thecoding portion of the human dopamine D₂ receptor gene is divided intoseven exons (FIG. 21, line b). Interestingly, we found that exon five is87-bp long and encodes the entire 29 amino acid sequence present in thecloned human pituitary receptor (FIGS. 18 and 21, line c). Analysis ofthe six introns revealed that each contains acceptor and donor sequencesthat conform to the GT/AG rule (20), as summarized in FIG. 23. Theapproximate sizes of the introns were based on the results of Southernblotting experiments (data not shown). When compared, the genomic andcDNA sequences were found to differ by only two silent transitions, oneat 939 (T to C in the gene) and the other at 957 (C to T in the gene).

Discussion

Several alternative hypotheses might account for the extra sequencepresent in our human pituitary D₂ receptor cDNA clone. One possibilityis that the human gene contains the extra 87 bases and that the rat genedoes not. Another is that both human and rat have two distinct geneswhich code for two different dopamine D₂ receptors. Finally, alternativesplicing of a single transcript could result in one mRNA and the otherwithout the 87 bases. In support of the latter hypothesis, we have shownthat there is probably only one human dopamine D₂ receptor gene, DRD2,and that the 87-bp sequence is contained on a distinct exon of thatgene. Furthermore, we have cloned a rat brain cDNA that contains the87-bp sequence (unpublished results). This sequence is highly similar tothat of the human cDNA and established that dopamine D₂ receptorscontaining the 29 residues are not unique to the human pituitary.

The human dopamine D₂ receptor expressed in L-hpitD₂Zem cells hasessentially the same drug binding profile as do rat striatum andL-RGB2Zem-1 membranes. Therefore, since the 29 amino acids do not affectbinding, they may be involved in other levels of receptor function. Forexample, the third cytoplasmic loop of the β₂-adrenergic receptor. hasbeen shown to be required for appropriate G-protein coupling (21).Therefore, one possibility is that this sequence may influence whetherdopamine D₂ receptor stimulation inhibits adenylyl cyclase, activatespotassium channel conductance or inhibits calcium mobilization (22).Another possibility is that the 29 amino acids differentiatepost-synaptic dopamine D₂ receptors from presynaptic autoreceptors (23).Since a computer search (VAX/Intelligenetics) failed to identify anothersequence of significant homology, we consider this sequence to be uniqueto the D₂ receptor.

The interruption of coding sequence by introns distinguishes the humandopamine D₂ receptor gene from most other members of the G-proteincoupled receptor gene family with the exception of the opsin genes (25).One significant observation is that the placement of two introns (Nos. 3and 5) in this human gene corresponds almost precisely to intronpositions conserved in bovine and Drosophila opsin genes (26, 27). Thesimplest interpretation of this finding is that their common ancestor, agene rougly one billion years old (28), contained these introns. Ourcharacterization of the gene structure also provides evidence that theexons encode recognizable elements of protein structure (29). Thatintrons are found following transmembrane segments II, III, and IV (SeeFIGS. 18 and 21) argues that the repeated structural motifscharacteristic of these receptors may have evolved by internalduplication. Furthermore, the presence of several introns within thethird cytoplasmic loop provides an explanation of the substantialvariation in length observed across the family (30).

The possibility of alternatively spliced dopamine D₂ receptdr mRNAsgiving rise to structurally distinct forms is exciting. The expressionof one form of the dopamine D₂ receptor mRNA or another represents alevel of control which may have implications with respect to humandisease.

Acknowledmements

We would like to thank Howard Goodman for discussion and review of themanuscript, Dee Yarozeski for manuscript preparation, and VickyRobertson, Nancy Kurkinen, and June Shiigi for the illustrations. D.K.G.holds a fellowship from NIH. This work was supported by NIH Grant Nos.Dk37231 and MH45614 and a grant from Cambridge NeuroScience Research,Inc., Cambridge, Mass., to O.C.

References

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What is claimed is:
 1. A method of screening a compound for binding to amammalian D2 dopamine receptor on the surface of cells expressing thereceptor, the method comprising the following steps: (a) transforming ahost cell with a recombinant expression construct encoding a mammalianD2 dopamine receptor having an amino acid sequence depicted in FIGS. 1Athrough 1E or in FIGS. 18A through 18H or an amino acid sequencedepicted in FIGS. 18A through 18H wherein amino acid residues 242through 270 are deleted therefrom and amino acid 271 is an aspartic acidresidue, wherein the cells of the transformed cell culture express thereceptor; and (b) assaying the transformed cell with the compound todetermine whether the compound binds to the receptor.
 2. A method ofscreening a compound for competitive binding to a mammalian D2 dopaminereceptor on the surface of cells expressing the receptor, the methodcomprising the following steps: (a) transforming a host cell with arecombinant expression construct encoding a mammalian D2 dopaminereceptor having an amino acid sequence depicted in FIGS. 1A through 1Eor in FIGS. 18A through 18H or an amino acid sequence depicted in FIGS.18A though 18H wherein amino acid residues 242 through 270 are deletedtherefrom and amino acid 271 is an aspartic acid residue, wherein thecells of the transformed cell culture express the receptor; (b) assayingthe transformed cell with the compound in the presence and in theabsence of an agonist for the receptor; and (c) determining whether thecompound competes with the agonist for binding to the receptor.
 3. Themethod of claim 2, wherein the compound is detectably-labeled.
 4. Themethod of claim 2, wherein the D2 dopamine receptor agonist isdetectably-labeled.
 5. The method of claim 2, wherein the compound thatcompetitively binds to the D2 dopamine receptor is quantitativelycharacterized by assaying the transformed cell culture with varyingamounts of the compound in the presence of a detectably-labeled D2dopamine receptor agonist and measuring the extent of competition withagonist binding thereby.
 6. A method of screening a compound todetermine if the compound is an agonist binding inhibitor of a mammalianD2 dopamine receptor on the surface of cells expressing the receptor,the method comprising the following steps: (a) transforming a host cellwith a recombinant expression construct encoding a mammalian D2 dopaminereceptor having an amino acid sequence depicted in FIGS. 1A through 1Eor in FIGS. 18A through 18H or an amino acid sequence depicted in FIGS.18A though 18H wherein amino acid residues 242 through 270 are deletedtherefrom and amino acid 271 is an aspartic acid residue, wherein thecells of the transformed cell culture express the receptor; and (b)assaying the transformed cell culture with the compound in the presenceand absence of a D2 dopamine receptor agonist to determine whether thecompound is capable of inhibiting agonist binding to receptor.
 7. Themethod of claim 6, wherein the compound is detectably-labeled.
 8. Themethod of claim 6, wherein the D2 dopamine receptor agonist isdetectably-labeled.
 9. The method of claim 6, wherein the compound thatinhibits D2 dopamine receptor agonist binding is quantitativelycharacterized by assaying the transformed cell culture with varyingamounts of the compound in the presence of a detectably-labeled receptorbinding agonist and measuring the extent of inhibition of agonistbinding thereby.